Sample records for cholecystokinin-2 receptor mediated

  1. Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors. (United States)

    Sanchez, Claire; Escrieut, Chantal; Clerc, Pascal; Gigoux, Véronique; Waser, Beatrice; Reubi, Jean Claude; Fourmy, Daniel


    The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

  2. Regulation of membrane cholecystokinin-2 receptor by agonists enables classification of partial agonists as biased agonists. (United States)

    Magnan, Rémi; Masri, Bernard; Escrieut, Chantal; Foucaud, Magali; Cordelier, Pierre; Fourmy, Daniel


    Given the importance of G-protein-coupled receptors as pharmacological targets in medicine, efforts directed at understanding the molecular mechanism by which pharmacological compounds regulate their presence at the cell surface is of paramount importance. In this context, using confocal microscopy and bioluminescence resonance energy transfer, we have investigated internalization and intracellular trafficking of the cholecystokinin-2 receptor (CCK2R) in response to both natural and synthetic ligands with different pharmacological features. We found that CCK and gastrin, which are full agonists on CCK2R-induced inositol phosphate production, rapidly and abundantly stimulate internalization. Internalized CCK2R did not rapidly recycle to plasma membrane but instead was directed to late endosomes/lysosomes. CCK2R endocytosis involves clathrin-coated pits and dynamin and high affinity and prolonged binding of β-arrestin1 or -2. Partial agonists and antagonists on CCK2R-induced inositol phosphate formation and ERK1/2 phosphorylation did not stimulate CCK2R internalization or β-arrestin recruitment to the CCK2R but blocked full agonist-induced internalization and β-arrestin recruitment. The extreme C-terminal region of the CCK2R (and more precisely phosphorylatable residues Ser(437)-Xaa(438)-Thr(439)-Thr(440)-Xaa(441)-Ser(442)-Thr(443)) were critical for β-arrestin recruitment. However, this region and β-arrestins were dispensable for CCK2R internalization. In conclusion, this study allowed us to classify the human CCK2R as a member of class B G-protein-coupled receptors with regard to its endocytosis features and identified biased agonists of the CCK2R. These new important insights will allow us to investigate the role of internalized CCK2R·β-arrestin complexes in cancers expressing this receptor and to develop new diagnosis and therapeutic strategies targeting this receptor.

  3. Transcriptomic and behavioural characterisation of a mouse model of burn pain identify the cholecystokinin 2 receptor as an analgesic target. (United States)

    Yin, Kathleen; Deuis, Jennifer R; Lewis, Richard J; Vetter, Irina


    Burn injury is a cause of significant mortality and morbidity worldwide and is frequently associated with severe and long-lasting pain that remains difficult to manage throughout recovery. We characterised a mouse model of burn-induced pain using pharmacological and transcriptomic approaches. Mechanical allodynia elicited by burn injury was partially reversed by meloxicam (5 mg/kg), gabapentin (100 mg/kg) and oxycodone (3 and 10 mg/kg), while thermal allodynia and gait abnormalities were only significantly improved by amitriptyline (3 mg/kg) and oxycodone (10 mg/kg). The need for relatively high opioid doses to elicit analgesia suggested a degree of opioid resistance, similar to that shown clinically in burn patients. We thus assessed the gene expression changes in dorsal root ganglion neurons and pathophysiological mechanisms underpinning burn injury-induced pain using a transcriptomic approach. Burn injury was associated with significantly increased expression of genes associated with axon guidance, neuropeptide signalling, behavioural defence response and extracellular signalling, confirming a mixed neuropathic and inflammatory aetiology. Notably, among the pain-related genes that were upregulated post-injury was the cholecystokinin 2 receptor (Cckbr), a G protein-coupled receptor known as a pain target involved in reducing opioid effectiveness. Indeed, the clinically used cholecystokinin receptor antagonist proglumide (30 mg/kg) was effective at reversing mechanical allodynia, with additional analgesia evident in combination with low-dose oxycodone (1 mg/kg), including significant reversal of thermal allodynia. These findings highlight the complex pathophysiological mechanisms underpinning burn injury-induced pain and suggest that cholecystokinin-2 receptor antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.

  4. Expression of cholecystokinin2-receptor in rat and human L cells and the stimulation of glucagon-like peptide-1 secretion by gastrin treatment. (United States)

    Cao, Yang; Cao, Xun; Liu, Xiao-Min


    Gastrin is a gastrointestinal hormone secreted by G cells. Hypergastrinemia can improve blood glucose and glycosylated hemoglobin levels. These positive effects are primarily due to the trophic effects of gastrin on β-cells. In recent years, many receptors that regulate secretion of glucagon-like peptide 1 (GLP-1) have been identified in enteroendocrine L cell lines. This led us to hypothesize that, in addition to the trophic effects of gastrin on β-cells, L cells also express cholecystokinin2-receptor (CCK2R), which may regulate GLP-1 secretion and have synergistic effects on glucose homeostasis. Our research provides a preliminary analysis of CCK2R expression and the stimulating effect of gastrin treatment on GLP-1 secretion in a human endocrine L cell line, using RT-PCR, Western blot, immunocytochemistry, and ELISA analyses. The expression of proglucagon and prohormone convertase 3, which regulate GLP-1 biosynthesis, were also analyzed by real-time PCR. Double immunofluorescence labeling was utilized to assess the intracellular localization of CCK2R and GLP-1 in L cells harvested from rat colon tissue. Our results showed that CCK2R was expressed in both the human L cell line and the rat L cells. We also showed that treatment with gastrin, a CCK2R agonist, stimulated the secretion of GLP-1, and that this effect was likely due to increased expression of proglucagon and PCSK1 (also known as prohormone convertase 3 (PC3 gene)). These results not only provide a basis for the role gastrin may play in intestinal L cells, and may also provide the basis for the development of a method of gastrin-mediated glycemic regulation.

  5. Cholecystokinin-2 receptor mediated gene expression in neuronal PC12 cells

    DEFF Research Database (Denmark)

    Hansen, Thomas v O; Borup, Rehannah; Marstrand, Troels;


    Cholecystokinin (CCK) is abundantly expressed in the CNS, in which it regulates feeding behavior and long-term memory. Moreover, CCK has been implicated in mental disorders, such as anxiety and schizophrenia. Despite its manifest physiological and pathophysiological role, the molecular targets...... could be identified. Comparison with forskolin- and nerve growth factor (NGF)-treated PC12 cells showed that CCK induced a separate set of target genes. Taken together, we propose that neuronal CCK may have a role in the regulation of the circadian rhythm, the metabolism of cerebral cholesterol...

  6. Natural sweetener agave inhibits gastric emptying in rats by a cholecystokinin-2- and glucagon like peptide-1 receptor-dependent mechanism. (United States)

    Bihter Gürler, E; Özbeyli, Dilek; Buzcu, Hülya; Bayraktar, Sezin; Carus, İrem; Dağ, Beyza; Geriş, Yasemin; Jeral, Seda; Yeğen, Berrak Ç


    Low-calorie sweeteners are considered to be beneficial in calorie control, but the impact of these sweeteners on gastric emptying is not well described. The purpose of this study was to compare the gastric emptying rate of agave nectar with those of glucose and fructose, and to evaluate the interaction of cholecystokinin (CCK)-1, CCK-2 and glucagon-like peptide-1 (GLP-1) receptors in agave-induced alterations in gastric emptying. Female Sprague-Dawley rats were fitted with gastric cannulas. Following the recovery, the gastric emptying rates of glucose, fructose and agave at 12.5%, 15% or 50% concentrations were measured and compared with that of saline. GLP-1 receptor antagonist exendin fragment 9-39 (30 μg kg(-1)), CCK-1 receptor antagonist devazepide (1 mg kg(-1)) or gastrin/CCK-2 receptor antagonist YM022 (1 mg kg(-1)) was injected subcutaneously 1 min before the emptying of glucose, fructose or agave at their 50% concentrations. When compared with saline emptying, gastric emptying of glucose was significantly delayed at its 25% and 50% concentrations, but the emptying of 12.5% glucose was not different from that of saline. Agave emptying, which was delayed with respect to saline emptying, was not altered by CCK-1 receptor blockade; but agave emptied from the stomach as rapidly as saline following the blockade of either CCK-2 or GLP-1 receptors. The findings demonstrate that the inhibitory effect of agave on gastric emptying is mediated by both CCK-2 and GLP-1 receptors, suggesting that natural sweeteners including agave may have satiating effects through the inhibition of gastric motility via enteroendocrine mechanisms.

  7. Regulation of Estrogen Receptor Nuclear Export by Ligand-Induced and p38-Mediated Receptor Phosphorylation


    Lee, Heehyoung; Bai, Wenlong


    Estrogen receptors are phosphoproteins which can be activated by ligands, kinase activators, or phosphatase inhibitors. Our previous study showed that p38 mitogen-activated protein kinase was involved in estrogen receptor activation by estrogens and MEKK1. Here, we report estrogen receptor-dependent p38 activation by estrogens in endometrial adenocarcinoma cells and in vitro and in vivo phosphorylation of the estrogen receptor α mediated through p38. The phosphorylation site was identified as...

  8. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    Directory of Open Access Journals (Sweden)

    Shanshan Wang


    Full Text Available 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.

  9. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications. (United States)

    D'Souza, Anisha A; Devarajan, Padma V


    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.

  10. Nonlinear pharmacokinetics of therapeutic proteins resulting from receptor mediated endocytosis. (United States)

    Krippendorff, Ben-Fillippo; Kuester, Katharina; Kloft, Charlotte; Huisinga, Wilhelm


    Receptor mediated endocytosis (RME) plays a major role in the disposition of therapeutic protein drugs in the body. It is suspected to be a major source of nonlinear pharmacokinetic behavior observed in clinical pharmacokinetic data. So far, mostly empirical or semi-mechanistic approaches have been used to represent RME. A thorough understanding of the impact of the properties of the drug and of the receptor system on the resulting nonlinear disposition is still missing, as is how to best represent RME in pharmacokinetic models. In this article, we present a detailed mechanistic model of RME that explicitly takes into account receptor binding and trafficking inside the cell and that is used to derive reduced models of RME which retain a mechanistic interpretation. We find that RME can be described by an extended Michaelis-Menten model that accounts for both the distribution and the elimination aspect of RME. If the amount of drug in the receptor system is negligible a standard Michaelis-Menten model is capable of describing the elimination by RME. Notably, a receptor system can efficiently eliminate drug from the extracellular space even if the total number of receptors is small. We find that drug elimination by RME can result in substantial nonlinear pharmacokinetics. The extent of nonlinearity is higher for drug/receptor systems with higher receptor availability at the membrane, or faster internalization and degradation of extracellular drug. Our approach is exemplified for the epidermal growth factor receptor system.

  11. Nuclear receptors : mediators and modifiers of inflammation-induced cholestasis

    NARCIS (Netherlands)

    Mulder, Jaap; Karpen, Saul J.; Tietge, Uwe J. F.; Kuipers, Folkert


    Inflammation-induced cholestasis (IIC) is a frequently occurring phenomenon. A central role in its pathogenesis is played by nuclear receptors (NRs). These ligand-activated transcription factors not only regulate basal expression of hepatobiliary transport systems, but also mediate adaptive response

  12. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes (United States)

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


    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

  13. Scavenger Receptor Mediates Systemic RNA Interference in Ticks


    Kyaw Min Aung; Damdinsuren Boldbaatar; Rika Umemiya-Shirafuji; Min Liao; Xuan Xuenan; Hiroshi Suzuki; Remil Linggatong Galay; Tetsuya Tanaka; Kozo Fujisaki


    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR ge...

  14. Receptor tyrosine phosphatase R-PTP-kappa mediates homophilic binding

    DEFF Research Database (Denmark)

    Sap, J; Jiang, Y P; Friedlander, D


    Receptor tyrosine phosphatases (R-PTPases) feature PTPase domains in the context of a receptor-like transmembrane topology. The R-PTPase R-PTP-kappa displays an extracellular domain composed of fibronectin type III motifs, a single immunoglobulin domain, as well as a recently defined MAM domain (Y.......-P. Jiang, H. Wang, P. D'Eustachio, J.M. Musacchio, J. Schlessinger, and J. Sap, Mol. Cell. Biol. 13:2942-2951, 1993). We report here that R-PTP-kappa can mediate homophilic intercellular interaction. Inducible expression of the R-PTP-kappa protein in heterologous cells results in formation of stable...... cellular aggregates strictly consisting of R-PTP-kappa-expressing cells. Moreover, the purified extracellular domain of R-PTP-kappa functions as a substrate for adhesion by cells expressing R-PTP-kappa and induces aggregation of coated synthetic beads. R-PTP-kappa-mediated intercellular adhesion does...

  15. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice. (United States)

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R


    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice.

  16. Purinergic Receptors: Key Mediators of HIV-1 infection and inflammation

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    Talia H Swartz


    Full Text Available Human immunodeficiency virus (HIV-1 causes a chronic infection that afflicts more than 38 million individuals worldwide. While the infection can be suppressed with potent anti-retroviral therapies, individuals infected with HIV have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets.

  17. Receptor-mediated choreography of life and death. (United States)

    Bhardwaj, Anjana; Aggarwal, Bharat B


    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  18. Of pheromones and kairomones: what receptors mediate innate emotional responses? (United States)

    Fortes-Marco, Lluis; Lanuza, Enrique; Martinez-Garcia, Fernando


    Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation.

  19. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

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    Fabio Cattaneo


    Full Text Available G protein-coupled receptors (GPCRs are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors

  20. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans. (United States)

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


    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.

  1. Receptor- and reactive intermediate-mediated mechanisms of teratogenesis. (United States)

    Wells, Peter G; Lee, Crystal J J; McCallum, Gordon P; Perstin, Julia; Harper, Patricia A


    Drugs and environmental chemicals can adversely alter the development of the fetus at critical periods during pregnancy, resulting in death, or in structural and functional birth defects in the surviving offspring. This process of teratogenesis may not be evident until a decade or more after birth. Postnatal functional abnormalities include deficits in brain function, a variety of metabolic diseases, and cancer. Due to the high degree of fetal cellular division and differentiation, and to differences from the adult in many biochemical pathways, the fetus is highly susceptible to teratogens, typically at low exposure levels that do not harm the mother. Insights into the mechanisms of teratogenesis come primarily from animal models and in vitro systems, and involve either receptor-mediated or reactive intermediate-mediated processes. Receptor-mediated mechanisms involving the reversible binding of xenobiotic substrates to a specific receptor are exemplified herein by the interaction of the environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or "dioxin") with the cytosolic aryl hydrocarbon receptor (AHR), which translocates to the nucleus and, in association with other proteins, binds to AH-responsive elements (AHREs) in numerous genes, initiating changes in gene transcription that can perturb development. Alternatively, many xenobiotics are bioactivated by fetal enzymes like the cytochromes P450 (CYPs) and prostaglandin H synthases (PHSs) to highly unstable electrophilic or free radical reactive intermediates. Electrophilic reactive intermediates can covalently (irreversibly) bind to and alter the function of essential cellular macromolecules (proteins, DNA), causing developmental anomalies. Free radical reactive intermediates can enhance the formation of reactive oxygen species (ROS), resulting in oxidative damage to cellular macromolecules and/or altered signal transduction. The teratogenicity of reactive intermediates is determined to a large extent

  2. Menthol inhibits 5-HT3 receptor-mediated currents. (United States)

    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


    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.

  3. P2X7 receptors mediate ischemic damage to oligodendrocytes. (United States)

    Domercq, Maria; Perez-Samartin, Alberto; Aparicio, David; Alberdi, Elena; Pampliega, Olatz; Matute, Carlos


    Brain ischemia leading to stroke is a major cause of disability in developed countries. Therapeutic strategies have most commonly focused on protecting neurons from ischemic damage. However, ischemic damage to white matter causes oligodendrocyte death, myelin disruption, and axon dysfunction, and it is partially mediated by glutamate excitotoxicity. We have previously demonstrated that oligodendrocytes express ionotropic purinergic receptors. The objective of this study was to investigate the role of purinergic signaling in white matter ischemia. We show that, in addition to glutamate, enhanced ATP signaling during ischemia is also deleterious to oligodendrocytes and myelin, and impairs white matter function. Thus, ischemic oligodendrocytes in culture display an inward current and cytosolic Ca(2+) overload, which is partially mediated by P2X7 receptors. Indeed, oligodendrocytes release ATP after oxygen and glucose deprivation through the opening of pannexin hemichannels. Consistently, ischemia-induced mitochondrial depolarization as well as oxidative stress culminating in cell death are partially reversed by P2X7 receptor antagonists, by the ATP degrading enzyme apyrase and by blockers of pannexin hemichannels. In turn, ischemic damage in isolated optic nerves, which share the properties of brain white matter, is greatly attenuated by all these drugs. Ultrastructural analysis and electrophysiological recordings demonstrated that P2X7 antagonists prevent ischemic damage to oligodendrocytes and myelin, and improved action potential recovery after ischemia. These data indicate that ATP released during ischemia and the subsequent activation of P2X7 receptor is critical to white matter demise during stroke and point to this receptor type as a therapeutic target to limit tissue damage in cerebrovascular diseases.

  4. Quantitation of the Contractile Response Mediated by Two Receptors: M2 and M3 Muscarinic Receptor-Mediated Contractions of Human Gastroesophageal Smooth MuscleS⃞ (United States)

    Braverman, Alan S.; Miller, Larry S.; Vegesna, Anil K.; Tiwana, Mansoor I.; Tallarida, Ronald J.; Ruggieri, Michael R.


    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M2 and M3 muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M2 receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M3 density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M2 and M3 receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M2 and M3 receptors to contraction is presented. Using this new method of analysis, it was found that the M2 muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M3 receptor predominantly mediates contraction. PMID:19126780

  5. Quantitation of the contractile response mediated by two receptors: M2 and M3 muscarinic receptor-mediated contractions of human gastroesophageal smooth muscle. (United States)

    Braverman, Alan S; Miller, Larry S; Vegesna, Anil K; Tiwana, Mansoor I; Tallarida, Ronald J; Ruggieri, Michael R


    Although muscarinic receptors are known to mediate tonic contraction of human gastrointestinal tract smooth muscle, the receptor subtypes that mediate the tonic contractions are not entirely clear. Whole human stomachs with attached esophagus were procured from organ transplant donors. Cholinergic contractile responses of clasp, sling, lower esophageal circular (LEC), midesophageal circular (MEC), and midesophageal longitudinal (MEL) muscle strips were determined. Sling fibers contracted greater than the other fibers. Total, M(2) and M(3) muscarinic receptor density was determined for each of these dissections by immunoprecipitation. M(2) receptor density is greatest in the sling fibers, followed by clasp, LEC, MEC, and then MEL, whereas M(3) density is greatest in LEC, followed by MEL, MEC, sling, and then clasp. The potency of subtype-selective antagonists to inhibit bethanechol-induced contraction was calculated by Schild analysis to determine which muscarinic receptor subtypes contribute to contraction. The results suggest both M(2) and M(3) receptors mediate contraction in clasp and sling fibers. Thus, this type of analysis in which multiple receptors mediate the contractile response is inappropriate, and an analysis method relating dual occupation of M(2) and M(3) receptors to contraction is presented. Using this new method of analysis, it was found that the M(2) muscarinic receptor plays a greater role in mediating contraction of clasp and sling fibers than in LEC, MEC, and MEL muscles in which the M(3) receptor predominantly mediates contraction.

  6. Estrogen receptor- and aryl hydrocarbon receptor- mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R. [Michigan State University, East Lansing, MI (USA). Dept. of Biochemistry


    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol- equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyp 1a1-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O- depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal- tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic response in vivo.

  7. Estrogen receptor- and aryl hydrocarbon receptor-mediated activities of a coal-tar creosote

    Energy Technology Data Exchange (ETDEWEB)

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R.


    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol-equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyplal-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O-depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal-tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic responses in vivo.

  8. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines (United States)

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


    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

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

    Directory of Open Access Journals (Sweden)

    Ashiq Hussain


    Full Text Available 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.

  10. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines. (United States)

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


    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.

  11. The type I interleukin-1 receptor mediates fever in the rat as shown by interleukin-1 receptor subtype selective ligands. (United States)

    Malinowsky, D; Chai, Z; Bristulf, J; Simoncsits, A; Bartfai, T


    The interleukin-1 (IL-1) system possesses two distinct receptors (type I and type II) which, together with the accessory protein, mediate a multitude of responses to IL-1 alpha and IL-1 beta, including fever. So far, no receptor subtype-specific ligands have been described. Since both types of IL-1 receptors occur in the thermoregulatory areas it was unclear which IL-1 receptor type mediates fever. We report here that for a series of deletion mutants of human recombinant IL-1 beta (hrIL-1 beta), the affinity of these ligands for the type I IL-1 receptor correlates with their efficacy to evoke the fever response (hrIL-1 beta > des-SND52-54 > des-QGE48-50 > des-I56). Thus, the results suggest that agonist occupancy of the type I IL-1 receptor is essential for IL-1 beta-mediated fever.

  12. Cytotoxicity mediated by human Fc receptors for IgG. (United States)

    Fanger, M W; Shen, L; Graziano, R F; Guyre, P M


    The Fc receptors for IgG(Fc gamma R) play a major role in the removal of antibody-coated infectious agents and may be important molecules for triggering cytotoxicity of tumor cells; they may also serve as an entry for infection of Fc gamma R-bearing cells by viral (including HIV and Dengue), and perhaps other infectious agents. Although central to immune defense, an understanding of the role of these Fc gamma R in cytotoxicity has been complicated in part by the presence of several biochemically distinct types of receptor that have different distributions, specificities, affinities and modes of activation for killing. The development of monoclonal antibodies specific for Fc gamma R on human leukocytes has established the existence of three distinct Fc gamma R and furthermore has helped clarify the function of each of these receptors. In this review, Michael Fanger and colleagues discuss the use of Fc gamma R-specific mAb and the hybridoma cell lines that produce them in examining the ability of each of these unique receptors to mediate killing of tumor and red cell targets. In particular, the use of self-directed hybridoma cells as a model of tumor-cell killing and of bi-specific antibodies to link target cells to effector cells through the different Fc gamma R is discussed. The results of these studies suggest that the ability of a given Fc gamma R to trigger killing is sometimes dependent on the type of Fc gamma R, but is also markedly influenced by the type of target cell and by the nature and state of activation of the effector cell.

  13. Ligand Receptor-Mediated Regulation of Growth in Plants. (United States)

    Haruta, Miyoshi; Sussman, Michael R


    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

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  15. NFAT regulates calcium-sensing receptor-mediated TNF production. (United States)

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


    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.

  16. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase. (United States)

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael


    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

  17. Discoidin Domain Receptor 1 Mediates Myosin-Dependent Collagen Contraction

    Directory of Open Access Journals (Sweden)

    Nuno M. Coelho


    Full Text Available Discoidin domain receptor 1 (DDR1 is a tyrosine kinase collagen adhesion receptor that mediates cell migration through association with non-muscle myosin IIA (NMIIA. Because DDR1 is implicated in cancer fibrosis, we hypothesized that DDR1 interacts with NMIIA to enable collagen compaction by traction forces. Mechanical splinting of rat dermal wounds increased DDR1 expression and collagen alignment. In periodontal ligament of DDR1 knockout mice, collagen mechanical reorganization was reduced >30%. Similarly, cultured cells with DDR1 knockdown or expressing kinase-deficient DDR1d showed 50% reduction of aligned collagen. Tractional remodeling of collagen was dependent on DDR1 clustering, activation, and interaction of the DDR1 C-terminal kinase domain with NMIIA filaments. Collagen remodeling by traction forces, DDR1 tyrosine phosphorylation, and myosin light chain phosphorylation were increased on stiff versus soft substrates. Thus, DDR1 clustering, activation, and interaction with NMIIA filaments enhance the collagen tractional remodeling that is important for collagen compaction in fibrosis.

  18. Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains



    Neurotensin receptor-1 (NTSR-1) is a G-protein coupled receptor (GPCR) that has been recently identified as a mediator of cancer progression. NTSR-1 and its endogenous ligand, neurotensin (NTS), are co-expressed in several breast cancer cell lines and breast cancer tumor samples. Based on our previously published study demonstrating that intact structured membrane microdomains (SMDs) are required for NTSR-1 mitogenic signaling, we hypothesized that regulated receptor palmitoylation is respons...

  19. Advance research in apoptosis mediating by death receptor

    Institute of Scientific and Technical Information of China (English)

    JI Yu-bin; LIU Hong-juan; JI Chen-feng; ZHANG He


    tissues, and is thought to play a role in tumor immune surveillance by NK, T cells and probably also cells of the innate immune system. The interaction between TNF and its ligand (TRAIL) recruits and activates caspases, TRAIL transmitting the signals through FADD, the probable mechanism is that:activated TRAIL combined with FADD through mutual recognition of Death domain, FADD combined MACH/FLICE(caspase-8), and then activates ICE/CED3 of caspase-8 and other proteins. The vast majority of cancer chemotherapy drug, mainly play a role through mitochondria-induced apoptosis pathway. TRAIL-induced apoptosis rely on the activation of caspase-8 and caspase-3. The mitochondrial membrane potential was reducted and the cytochrome C was released, leading to no damage of mitochondrial integrity after DNA cracking. Caspase is a specific type of acid cysteine protease, in apoptosis directly involved in the process of implementing the early start of apoptosis, signal transduction and apoptosis of late. Caspase-8 plays a key role in the death receptor-mediated apoptosis.

  20. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    Energy Technology Data Exchange (ETDEWEB)

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D' Avis, J.C.; Wartofsky, L. (Walter Reed Army Medical Center, Washington, DC (USA))


    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  1. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis (United States)

    Gonçalves-Carneiro, Daniel; McKeating, Jane A.


    ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between

  2. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain. (United States)

    Schlicker, E; Werthwein, S; Zentner, J


    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.

  3. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction. (United States)

    Braverman, Alan S; Tallarida, Ronald J; Ruggieri, Michael R


    M(3) muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M(2) receptors participate in contraction because M(3)-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M(2)-selective antagonist methoctramine in the denervated bladder is consistent with M(3) receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M(2) receptor and one by the M(3) receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M(2) and M(3) receptors can induce contraction. In the denervated bladder, the M(2) and the M(3) receptors interact in a facilitatory manner to mediate contraction.

  4. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction (United States)



    M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction. PMID:12185001

  5. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking (United States)

    Cole, Sindy; McNally, Gavan P.


    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  6. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals (United States)

    Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns (PAMPs) and endogenous molecules...

  7. Secretory phospholipase A2-mediated neuronal cell death involves glutamate ionotropic receptors

    DEFF Research Database (Denmark)

    de Turco, Elena B; Diemer, Nils Henrik; Bazan, Nicolas G


    To define the significance of glutamate ionotropic receptors in sPLA -mediated neuronal cell death we used the NMDA receptor antagonist MK-801 and the AMPA receptor antagonist PNQX. In primary neuronal cell cultures both MK-801 and PNQX inhibited sPLA - and glutamate-induced neuronal death. [ H...... neuronal cell death. We conclude that glutamatergic synaptic activity modulates sPLA -induced neuronal cell death....

  8. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids


    Chen Wang; Yi Liu; Ji-Min Cao


    Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs) are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G prot...

  9. Glutamate mediates the function of melanocortin receptor 4 on sim1 neurons in body weight regulation (United States)

    The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing ...


    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  11. Tracking Progesterone Receptor-Mediated Actions in Breast Cancer



    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 t...

  12. Quantifying Rosette Formation Mediated by Receptor-ligand Interactions

    Institute of Scientific and Technical Information of China (English)


    1 IntroductionRosetting is a simple assay for specific cell-cell adhesion, in which receptor- (or ligand-) coated RBCs form the rosettes with ligand- (or receptor-) expressed nucleated cells~([1]). Although routinely used by immunologists to examine the functionality of the interacting receptors and ligands, however, it has not been regarded as a quantitative method, as the measured rosette fraction has not been quantitatively related to the underlying molecular properties.Recently, we have solved probabili...

  13. Melanocortin MC(4) receptor-mediated feeding and grooming in rodents. (United States)

    Mul, Joram D; Spruijt, Berry M; Brakkee, Jan H; Adan, Roger A H


    Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced.

  14. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor. (United States)

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas


    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  15. Aryl hydrocarbon receptor mediates benzene-induced hematotoxicity. (United States)

    Yoon, Byung-Il; Hirabayashi, Yoko; Kawasaki, Yasushi; Kodama, Yukio; Kaneko, Toyozo; Kanno, Jun; Kim, Dae-Yong; Fujii-Kuriyama, Yoshiaki; Inoue, Tohru


    Benzene can induce hematotoxicity and leukemia in humans and mice. Since a review of the literature shows that the CYP2E1 knockout mouse is not known to possess any benzene toxicity, the metabolism of benzene by CYP2E1 in the liver is regarded to be prerequisite for its cytotoxicity and genotoxicity, although the mechanism is not fully understood yet. Because it was found some years ago that benzene was also a substrate for CYP1A1, we investigated the involvement of the aryl hydrocarbon receptor (AhR) in benzene hematotoxicity using AhR wild-type (AhR(+/+)), heterozygous (AhR(+/-)), and homozygous (AhR(-/-)) male mice. Interestingly, following a 2-week inhalation of 300 ppm benzene (a potent dose for leukemogenicity), no hematotoxicity was induced in AhR(-/-) mice. Further, there were no changes in cellularity of peripheral blood and bone marrow (BM), nor in levels of granulocyte-macrophage colony-forming units in BM. This lack of hematotoxicity was associated with the lack of p21 overexpression, which was regularly seen in the wild-type mice following benzene inhalation. Combined treatment with two major benzene metabolites, phenol and hydroquinone, induced hemopoietic toxicity, although it was not known whether this happened due to a surprising lack of expression of CYP2E1 by AhR knockout, or due to a lack of other AhR-mediated CYP enzymes, including 1A1 (i.e., a possible alternative pathway of benzene metabolism). The former possibility, evaluated in the present study, failed to show a significant relationship between AhR and the expression of CYP2E1. Furthermore, a subsequent evaluation of AhR expression after benzene inhalation tended to show higher but less significant expression in the liver, and none in the BM, compared with sham control. Although this study failed to identify the more likely of the above-mentioned two possibilities, the study using AhR knockout mice on benzene inhalation presents the unique possibility that the benzene toxicity may be

  16. Cysteinyl leukotriene receptor 1 partially mediates brain cryoinjury in mice

    Institute of Scientific and Technical Information of China (English)

    Qian DING; San-hua FANG; Yu ZHOU; Li-hui ZHANG; Wei-ping ZHANG; Zhong CHEN; Er-qing WEI


    Aim: To determine whether the cysteinyl leukotriene receptor 1 (CysLT1 receptor) modulates brain cryoinjury and whether the CysLT1 receptor antagonist pranlukast exerts a time-dependent protective effect on cryoinjury in mice. Methods: Brain cryoinjury was induced by applying a liquid nitrogen-cooled metal probe to the surface of the skull for 30 s. Brain lesion, neuron density, and endogenous IgG exudation were observed 24 h after cryoinjury. Transcription and the expression of the CysLT1 receptor were detected by RT-PCR and immunoblotting, and the localization of the receptor protein by double immunofluorescence. Results: The mRNA and protein expressions of the CysLT1 receptor were upregulated in the brain 6-24 h after cryoinjury, and the CysLT1 receptor protein was primarily local-ized in the neurons, not in the astrocytes or microglia. Pre-injury treatments with multi-doses and a single dose of pranlukast (0.1 mg/kg) attenuated cryoinjury; postinjury single dose (0.1 mg/kg) at 30 min (not 1 h) after cryoinjury was also effective. Conclusion: The CysLT1 receptor modulates cryoinjury in mice at least partly, and postinjury treatment with its antagonist pranlukast exerts the protec-tive effect with a therapeutic window of 30 min.

  17. Downregulation of adenosine and P2X receptor-mediated cardiovascular responses in heart failure rats

    DEFF Research Database (Denmark)

    Zhao, Xin; Sun, X Y; Erlinge, D;


    Neurohormonal changes in congestive heart failure (CHF) include an enhanced peripheral sympathetic nerve activity which results in increased release of noradrenaline, neuropeptide Y and ATP. To examine if such changes in CHF would modulate peripheral pre- and postsynaptic receptors of ATP and its...... effects mediated by the endothelial P2Y receptors are unaffected in CHF. Moreover, the adenosine-mediated inhibitory effects on heart rate and blood pressure were also attenuated in the CHF rats. The most important changes in adenosine and P2-receptor function induced by ischaemic CHF were the reduced...... pressor effect mediated by the P2X receptor and the increased heart rate due to an attenuated inhibitory effect of adenosine....

  18. Receptor mechanisms of PAF mediated lymphatic constriction in the canine forelimb

    Directory of Open Access Journals (Sweden)

    D. E. Dobbins


    Full Text Available Platelet activating factor (PAF is a potent inflammatory lipid. In this study we assessed the ability of PAF to impact lymphatic vessel function by altering prenodal lymphatic resistance. Intralymphatic PAF (7.47 × 10−6, 7.47 × 10−5 and 7.47 × 10−4 M increased lymphatic perfusion pressure at the two highest infusion rates. PAF mediated lymphatic constriction was not altered by the intra-arterial infusion of phentolamine but was blocked by the intra-arterial infusion of the PAF receptor antagonist WEB 2170. These data indicate that in addition to PAF's effects on microvascular permeability, this agent may also impact the ability of the lymphatics to transport fluid through alterations in lymphatic smooth muscle tone. PAF mediated lymphatic constriction is not mediated by α-receptors but rather through PAF receptor mediated mechanism.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Yuan, E-mail:; Cardell, Lars-Olaf


    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

  20. Interleukin-8-mediated heterologous receptor internalization provides resistance to HIV-1 infectivity. Role of signal strength and receptor desensitization. (United States)

    Richardson, Ricardo M; Tokunaga, Kenzo; Marjoram, Robin; Sata, Tetsutaro; Snyderman, Ralph


    Human immunodeficiency virus type 1 (HIV-1) entry into CD4(+) cells requires the chemokine receptors CCR5 or CXCR4 as co-fusion receptors. We have previously demonstrated that chemokine receptors are capable of cross-regulating the functions of each other and, thus, affecting cellular responsiveness at the site of infection. To investigate the effects of chemokine receptor cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat basophilic leukemia (RBL-2H3) cell lines co-expressing the interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and cross-desensitized their responsiveness to RANTES (regulated on activation normal T cell expressed and secreted) (CCL5) and stromal derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation internalized CCR5 but not CXCR4 despite cross-phosphorylation of both. IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated virus entry into MAGIC5 cells. A tail-deleted mutant of CXCR1, DeltaCXCR1, produced greater signals upon activation (Ca(2+) mobilization and phosphoinositide hydrolysis) and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein kinase C inhibitor staurosporine prevented phosphorylation and internalization of the receptors by CXCR1 activation. Taken together, these results indicate that chemokine receptor-mediated HIV-1 cell infection is blocked by receptor internalization but not desensitization alone. Thus, activation of chemokine receptors unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the infection and propagation of HIV-1. Since DeltaCXCR1, but not CXCR1, cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the data indicate the importance of the signal strength of a receptor and, as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.

  1. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado


    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  2. Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake.

    Directory of Open Access Journals (Sweden)

    Míriam Rodríguez-Vázquez


    Full Text Available Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP. We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them.

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

    Directory of Open Access Journals (Sweden)

    Daria eGuseva


    Full Text Available 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.

  4. Cellular mechanisms of the 5-HT7 receptor-mediated signaling. (United States)

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni


    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.

  5. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids

    Directory of Open Access Journals (Sweden)

    Chen Wang


    Full Text Available Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30 for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A and zinc transporter member 9 (ZIP9 for androgen, and trace amine associated receptor 1 (TAAR1 for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid, the structural basis of steroids and GPCRs’ interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  6. Agonist-mediated assembly of the crustacean methyl farnesoate receptor (United States)

    Kakaley, Elizabeth K. Medlock; Wang, Helen Y.; LeBlanc, Gerald A.


    The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

  7. An intracellular traffic jam: Fc receptor-mediated transport of immunoglobulin G. (United States)

    Tesar, Devin B; Björkman, Pamela J


    Recent advances in imaging techniques along with more powerful in vitro and in vivo models of receptor-mediated ligand transport are facilitating advances in our understanding of how cells efficiently direct receptors and their cargo to target destinations within the cytoplasm and at the plasma membrane. Specifically, light and 3D electron microscopy studies examining the trafficking behavior of the neonatal Fc receptor (FcRn), a transport receptor for immunoglobulin G (IgG), have given us new insights into the dynamic interplay between the structural components of the cytosolic trafficking machinery, its protein regulators, and the receptors it directs to various locations within the cell. These studies build upon previous biochemical characterizations of FcRn transport and are allowing us to begin formulation of a more complete model for the intracellular trafficking of receptor-ligand complexes.

  8. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling. (United States)

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen


    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  9. Nucleus accumbens μ-opioid receptors mediate social reward. (United States)

    Trezza, Viviana; Damsteegt, Ruth; Achterberg, E J Marijke; Vanderschuren, Louk J M J


    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

  10. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    Institute of Scientific and Technical Information of China (English)



    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  11. Upregulation of endothelin ETB receptor-mediated vasoconstriction in rat coronary artery after organ culture

    DEFF Research Database (Denmark)

    Eskesen, Karen; Edvinsson, Lars


    The aim of this study was to examine if endothelin ET(B) receptor-mediated contraction occurred in isolated segments of rat coronary arteries during organ culture. Presence of contractile endothelin ET(B) receptors was studied by measuring the change in isometric tension in rings of left anterior...... descending coronary arteries isolated from hearts of rats as response to application of the selective endothelin ET(B) receptor agonist, Sarafotoxin 6c and endothelin-1. In segments cultured 1 day in serum free Dulbecco's Modified Eagle's Medium, Sarafotoxin 6c induced a concentration dependent contraction......(+)-solution was not modified after 1 day in culture medium. The experiments indicate that organ culture of rat coronary arteries upregulate endothelin ET(B) receptor-mediated contraction by inducing synthesis of new protein....

  12. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission. (United States)

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L


    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  13. Experimental Cannabinoid 2 Receptor-Mediated Immune Modulation in Sepsis

    Directory of Open Access Journals (Sweden)

    J. Sardinha


    Full Text Available Sepsis is a complex condition that results from a dysregulated immune system in response to a systemic infection. Current treatments lack effectiveness in reducing the incidence and mortality associated with this disease. The endocannabinoid system offers great promise in managing sepsis pathogenesis due to its unique characteristics. The present study explored the effect of modulating the CB2 receptor pathway in an acute sepsis mouse model. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS in mice and intestinal microcirculation was assessed through intravital microscopy. We found that HU308 (CB2 receptor agonist reduced the number of adherent leukocytes in submucosal venules but did not restore muscular and mucosal villi FCD in endotoxemic mice. AM630 (CB2 receptor antagonist maintained the level of adherent leukocytes induced by LPS but further reduced muscular and mucosal villi FCD. URB597 (FAAH inhibitor and JZL184 (MAGL inhibitor both reduced the number of adherent leukocytes in submucosal venules but did not restore the mucosal villi FCD. Using various compounds we have shown different mechanisms of activating CB2 receptors to reduce leukocyte endothelial interactions in order to prevent further inflammatory damage during sepsis.

  14. Receptor targeting of hemoglobin mediated by the haptoglobins

    DEFF Research Database (Denmark)

    Nielsen, Marianne Jensby; Moestrup, Søren Kragh


    Haptoglobin, the haptoglobin-hemoglobin receptor CD163, and the heme oxygenase-1 are proteins with a well-established function in the clearance and metabolism of "free" hemoglobin released during intravascular hemolysis. This scavenging system counteracts the potentially harmful oxidative and NO-...

  15. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    Energy Technology Data Exchange (ETDEWEB)

    Magno, Aaron L. [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ingley, Evan [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Brown, Suzanne J. [Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Conigrave, Arthur D. [School of Molecular Bioscience, University of Sydney, New South Wales 2000 (Australia); Ratajczak, Thomas [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia); Ward, Bryan K., E-mail: [Western Australian Institute for Medical Research and Centre for Medical Research, University of Western Australia, Nedlands, Western Australia 6009 (Australia); Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Hospital Avenue, Nedlands, Western Australia 6009 (Australia)


    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  16. Multiple Novel Signals Mediate Thyroid Hormone Receptor Nuclear Import and Export*


    Mavinakere, Manohara S.; Powers, Jeremy M.; Subramanian, Kelly S.; Roggero, Vincent R.; Allison, Lizabeth A.


    Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we ...

  17. Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways


    Schlenz, Heike; Kummer, Wolfgang; Jositsch, Gitte; Wess, Jürgen; Krasteva, Gabriela


    Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well...

  18. Alpha-synuclein promotes clathrin-mediated endocytosis of NMDA receptors in dopaminergic cells

    Institute of Scientific and Technical Information of China (English)

    Shun Yu; Furong Cheng; Xin Li; Yaohua Li; Tao Wang; Guangwei Liu; Andrius Baskys


    Loss of dopaminergic i a compensatory increase in nput to the striatum associated with Parkinson' s disease brings about glutamate release onto the dopaminergic cell bodies in the substantia nigra pars compacta (SNpc)[1] Glutamate over-activation of NMDA receptors on these cells can cause excitotoxicity and contribute to their further loss. NMDA receptor-mediated neuronal death is reduced by group I mGluR-mediated up-regulation of endocytosis protein RAB5B[2.3] Among proteins shown to interact with RAB5 proteins is a-synuclein

  19. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro (United States)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki


    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  20. The Role of Cgrp-Receptor Component Protein (Rcp) in Cgrp-Mediated Signal Transduction


    Prado, M.A.; B. Evans-Bain; Santi, S. L.; Dickerson, I M


    The calcitonin gene-related peptide (CGRP)-receptor component protein (RCP) is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as...

  1. Effects of gamma-aminobutyric acid receptors on muscarinic receptor-mediated free calcium ion levels in the facial nucleus following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Guangfeng Jiang; Dawei Sun; Rui Zhou; Fugao Zhu; Yanqing Wang; Xiuming Wan; Banghua Liu


    Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B (GABAB) and C (GABAC) receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.

  2. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    Directory of Open Access Journals (Sweden)

    Anne-Christine Field

    Full Text Available Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  3. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses

    DEFF Research Database (Denmark)

    Tkaczyk, Christine; Jensen, Bettina M; Iwaki, Shoko


    differentially activate multiple signaling pathways within the mast cells required for the generation and/or release of inflammatory mediators. Thus, the composition of the suite of mediators released and the physiologic ramifications of these responses are dependent on the stimuli and the microenvironment...... activation. The exact interconnections between the signaling pathways initiated by the surface receptors described in this article remain to be completely worked out; thus, this remains a topic for future investigation....

  4. P2 receptor-mediated signaling in mast cell biology. (United States)

    Bulanova, Elena; Bulfone-Paus, Silvia


    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  5. Androgen Receptor-Mediated Escape Mechanisms from Androgen Ablation Therapy (United States)


    of CAG repeats in the Machado-Joseph disease , spinocerebellar ataxia type 1 and androgen receptor genes. Hum. Mol. Genet. 4, 1585-1590. Rundlett, S . E... diseases such as Huntington disease and spinal and bulbar muscular atro- phy, which is commonly called Kennedy’s disease . This finding has been attributed...STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author( s ) and

  6. P2 receptor-mediated signaling in mast cell biology


    Bulanova, Elena; Bulfone-Paus, Silvia


    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of ...

  7. Taste Receptors Mediate Sinonasal Immunity and Respiratory Disease (United States)

    Douglas, Jennifer E.; Cohen, Noam A.


    The bitter taste receptor T2R38 has been shown to play a role in the pathogenesis of chronic rhinosinusitis (CRS), where the receptor functions to enhance upper respiratory innate immunity through a triad of beneficial immune responses. Individuals with a functional version of T2R38 are tasters for the bitter compound phenylthiocarbamide (PTC) and exhibit an anti-microbial response in the upper airway to certain invading pathogens, while those individuals with a non-functional version of the receptor are PTC non-tasters and lack this beneficial response. The clinical ramifications are significant, with the non-taster genotype being an independent risk factor for CRS requiring surgery, poor quality-of-life (QOL) improvements post-operatively, and decreased rhinologic QOL in patients with cystic fibrosis. Furthermore, indirect evidence suggests that non-tasters also have a larger burden of biofilm formation. This new data may influence the clinical management of patients with infectious conditions affecting the upper respiratory tract and possibly at other mucosal sites throughout the body. PMID:28218655

  8. Neurotransmitter receptor-mediated signaling pathways as modulators of carcinogenesis. (United States)

    Schuller, Hildegard M


    The autonomic nervous system with its two antagonistic branches, the sympathicus and the parasympathicus, regulates the activities of all body functions that are not under voluntary control. While the autonomic regulation of organ functions has been extensively studied, little attention has been given to the potential role of neurohumoral transmission at the cellular level in the development of cancer. Studies conducted by our laboratory first showed that binding of the parasympathetic neurotransmitter, acetylcholine, as well as nicotine or its nitrosated cancer-causing derivative, NNK, to nicotinic acetylcholine receptors comprised of alpha7 subunits activated a mitogenic signal transduction pathway in normal and neoplastic pulmonary neuroendocrine cells. On the other hand, beta-adrenergic receptors (Beta-ARs), which transmit signals initiated by binding of the catecholamine neurotransmitters of the sympathicus, were identified by our laboratory as important regulators of cell proliferation in cell lines derived from human adenocarcinomas of the lungs, pancreas, and breast. The tobacco-specific carcinogen NNK bound with high affinity to Beta1- and Beta2-ARs, thus activating cAMP, protein kinase A, and the transcription factor CREB. Collectively, neurotransmitter receptors of the nicotinic and Beta-adrenergic families appear to regulate cellular functions essential for the development and survival of the most common human cancers.

  9. Taste Receptors Mediate Sinonasal Immunity and Respiratory Disease

    Directory of Open Access Journals (Sweden)

    Jennifer E. Douglas


    Full Text Available The bitter taste receptor T2R38 has been shown to play a role in the pathogenesis of chronic rhinosinusitis (CRS, where the receptor functions to enhance upper respiratory innate immunity through a triad of beneficial immune responses. Individuals with a functional version of T2R38 are tasters for the bitter compound phenylthiocarbamide (PTC and exhibit an anti-microbial response in the upper airway to certain invading pathogens, while those individuals with a non-functional version of the receptor are PTC non-tasters and lack this beneficial response. The clinical ramifications are significant, with the non-taster genotype being an independent risk factor for CRS requiring surgery, poor quality-of-life (QOL improvements post-operatively, and decreased rhinologic QOL in patients with cystic fibrosis. Furthermore, indirect evidence suggests that non-tasters also have a larger burden of biofilm formation. This new data may influence the clinical management of patients with infectious conditions affecting the upper respiratory tract and possibly at other mucosal sites throughout the body.

  10. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms. (United States)

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


    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).

  11. Isolated NMDA receptor-mediated synaptic responses express both LTP and LTD. (United States)

    Xie, X; Berger, T W; Barrionuevo, G


    1. The possibility of use-dependent, long-lasting modifications of pharmacologically isolated N-methyl-D-aspartate (NMDA) receptor-mediated synaptic transmission was examined by intracellular recordings from granule cells of the hippocampal dentate gyrus in vitro. In the presence of the non-NMDA receptor antagonist 6-cyano-7-nitroquinaxaline-2,3-dione (CNQX, 10 microM) robust, long-term potentiation (LTP) of NMDA receptor-mediated synaptic potentials was induced by brief, high (50 Hz) and lower (10 Hz) frequency tetanic stimuli of glutamatergic afferents (60 +/- 6%, n = 8, P less than 0.001 and 43 +/- 12%, n = 3, P less than 0.05, respectively). 2. Hyperpolarization of granule cell membrane potential to -100 mV during 50-Hz tetanic stimuli reversibly blocked the induction of LTP (-6 +/- 2%, n = 6, P greater than 0.05) indicating that simultaneous activation of pre- and postsynaptic elements is a prerequisite for potentiation of NMDA receptor-mediated synaptic transmission. In contrast, hyperpolarization of the granule cell membrane potential to -100 mV during 10-Hz tetanic stimuli resulted in long-term depression (LTD) of NMDA receptor-mediated synaptic potentials (-34 +/- 8%, n = 8, P less than 0.01). 3. We also studied the role of [Ca2+]i in the induction of LTP and LTD of NMDA receptor-mediated synaptic responses. Before tetanization, [Ca2+]i was buffered by iontophoretic injections of bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). BAPTA completely blocked the induction of LTP (3 +/- 5%, n = 13) and partially blocked LTD (-14.8 +/- 6%, n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

  12. (-)-Stepholodine induced enhancement of cardiac muscle contractions mediated by D1 receptors

    Institute of Scientific and Technical Information of China (English)

    ZHOU Shu-yuan; LIU Zheng; HU Hui-sheng; SHI Zhen; CHEN Long


    Objective To investigate the effect of (-)-Stepholidine (SPD) on enhancing D1 receptor mediated contraction of cardiac muscle in isolated rat heart and to examine whether SPD has a direct effect on the heart dopamine D1 receptors. SPD an active ingredient of the Chinese herb Stephania intermedia, binds to dopamine D1 and D2 like receptors. Biochemical, electrophysiological and behavioural experiments have provided strong evidence that SPD is both a D(1/5) agonist and a D(2/4) antagonist, which could indicate unique antipsychotic properties. Methods Normal adult rat working hearts were isolated by Langendorff technique. Results SPD significantly increased the cardiac muscle contraction in a dose-dependent manner. The selective D1 dopamine receptor antagonist SCH23390 (1 μM) blocked the SPD induced heart contraction, however, neither the β-receptor antagonist propranolol (1 μM) nor the α1-receptor antagonist prazosin (1 μM) had any effect on blocking SPD induced heart contractions. Moreover, the L-type Ca2+ channel inhibitor nimodipine (1 μM) completely blocked the effect of SPD on cardiac muscle contraction. Conclusions SPD show the effect on enhancing contraction of isolated rat heart through activating L-type Ca2+ channel mediated by heart D1 receptors.

  13. Possible Implication of Fcγ Receptor-Mediated Trogocytosis in Susceptibility to Systemic Autoimmune Disease

    Directory of Open Access Journals (Sweden)

    Sakiko Masuda


    Full Text Available Leukocytes can “gnaw away” the plasma membrane of other cells. This phenomenon, called trogocytosis, occurs subsequent to cell-to-cell adhesion. Currently, two mechanisms of trogocytosis, adhesion molecule-mediated trogocytosis and Fcγ receptor-(FcγR- mediated trogocytosis, have been identified. In our earlier study, we established an in vitro model of FcγR-mediated trogocytosis, namely, CD8 translocation model from T cells to neutrophils. By using this model, we demonstrated that the molecules transferred to neutrophils via FcγR-mediated trogocytosis were taken into the cytoplasm immediately. This result suggests that the chance of molecules transferred via FcγR-mediated trogocytosis to play a role on the cell surface could be time-limited. Thus, we consider the physiological role of FcγR-mediated trogocytosis as a means to remove antibodies (Abs that bind with self-molecules rather than to extract molecules from other cells. This concept means that FcγR-mediated trogocytosis can be a defense mechanism to Ab-mediated autoimmune response. Moreover, the activity of FcγR-mediated trogocytosis was revealed to be parallel to the endocytotic activity of neutrophils, which was critically related to the susceptibility to systemic autoimmune diseases. The collective findings suggest that FcγR-mediated trogocytosis could physiologically play a role in removal of Abs bound to self-antigens and prevent autoimmune diseases.

  14. Extranuclear Signaling Effects Mediated by the Estrogen Receptor (United States)


    Examination of the αCaMKII amino acid sequence revealed that it contains a nuclear receptor interaction motif, or NR box, within the CaM-binding region...inhibition of Pyk2 with salicylate disrupted the ability of E2 to induce the phosphorylation of ERK1/2 without affecting αCaMKII activity (data not...Bottner, M, Rune, GM (2006) Oestrogen synthesis in the hippocampus: role in axon outgrowth. J Neuroendocrinol 18:847-856. 14 Walf A, Frye CA (2007

  15. Lysosphingolipid receptor-mediated diuresis and natriuresis in anaesthetized rats. (United States)

    Bischoff, A; Meyer Zu Heringdorf, D; Jakobs, K H; Michel, M C


    Lysosphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act on specific G-protein-coupled receptors. Since SPP and SPPC cause renal vasoconstriction, we have investigated their effects on urine and electrolyte excretion in anaesthetized rats. Infusion of SPP (1 - 30 microg kg(-1) min(-1)) for up to 120 min dose-dependently but transiently (peak after 15 min, disappearance after 60 min) reduced renal blood flow without altering endogenous creatinine clearance. Nevertheless, infusion of SPP increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. These tubular lysosphingolipid effects developed more slowly (maximum after 60 - 90 min) and also abated more slowly upon lysosphingolipid washout than the renovascular effects. Infusion of SPPC, sphingosine and glucopsychosine (3 - 30 microg kg(-1) min(-1) each) caused little if any alterations in renal blood flow but also increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. Pretreatment with pertussis toxin (10 microg kg(-1) 3 days before the acute experiment) abolished the renovascular and tubular effects of 30 microg kg(-1) min(-1) SPP. These findings suggest that lysosphingolipids are a hitherto unrecognized class of endogenous modulators of renal function. SPP affects renovascular tone and tubular function via receptors coupled to G(i)-type G-proteins. SPPC, sphingosine and glucopsychosine mimic only the tubular effects of SPP, and hence may act on distinct sites.

  16. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    DEFF Research Database (Denmark)

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren


    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  17. Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture

    Directory of Open Access Journals (Sweden)

    John L. McDonald


    Full Text Available Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1, a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture.

  18. Glucocorticoid receptor, but not mineralocorticoid receptor, mediates cortisol regulation of epidermal ionocyte development and ion transport in zebrafish (danio rerio.

    Directory of Open Access Journals (Sweden)

    Shelly Abad Cruz

    Full Text Available Cortisol is the major endogenous glucocorticoid (GC both in human and fish, mediated by corticosteroid receptors. Due to the absence of aldosterone production in teleost fish, cortisol is also traditionally accepted to function as mineralocorticoid (MC; but whether it acts through the glucocorticoid receptor (GR or the mineralocorticoid receptor (MR remains a subject of debate. Here, we used loss-of-function and rescue assays to determine whether cortisol affects zebrafish epidermal ionocyte development and function via the GR and/or the MR. GR knockdown morphants displayed a significant decrease in the major ionocytes, namely Na(+-K(+-ATPase-rich cells (NaRCs and H(+-ATPase-rich cells (HRCs, as well as other cells, including epidermal stem cells (ESCs, keratinocytes, and mucus cells; conversely, cell numbers were unaffected in MR knockdown morphants. In agreement, GR morphants, but not MR morphants, exhibited decreased NaRC-mediated Ca(2+ uptake and HRC-mediated H(+ secretion. Rescue via GR capped mRNA injection or exogenous cortisol incubation normalized the number of epidermal ionocytes in GR morphants. We also provide evidence for GR localization in epidermal cells. At the transcript level, GR mRNA is ubiquitously expressed in gill sections and present in both NaRCs and HRCs, supporting the knockdown and functional assay results in embryo. Altogether, we have provided solid molecular evidence that GR is indeed present on ionocytes, where it mediates the effects of cortisol on ionocyte development and function. Hence, cortisol-GR axis performs the roles of both GC and MC in zebrafish skin and gills.

  19. Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses. (United States)

    Hayashi, Chie; Madrigal, Andres G; Liu, Xinyan; Ukai, Takashi; Goswami, Sulip; Gudino, Cynthia V; Gibson, Frank C; Genco, Caroline A


    Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotein E-deficient (ApoE(-/-)) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-gamma and the pro-inflammatory cytokines IL-1 beta, IL-6 and tumor necrosis factor-alpha in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2(-/-)) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen.

  20. Alteration of airway responsiveness mediated by receptors in ovalbumin-induced asthmatic E3 rats

    Institute of Scientific and Technical Information of China (English)

    Jing-wen LONG; Xu-dong YANG; Lei CAO; She-min LU; Yong-xiao CAO


    Aim:Airway hyperresponsiveness is a constant feature of asthma.The aim of the present study was to investigate airway hyperreactivity mediated by contractile and dilative receptors in an ovalbumin (OVA)-induced model of rat asthma.Methods:Asthmatic E3 rats were prepared by intraperitoneal injection with OVA/aluminum hydroxide and then challenged with intranasal instillation of OVA-PBS two weeks later.The myograph method was used to measure the responses of constriction and dilatation in the trachea,main bronchi and lobar bronchi.Results:In asthmatic E3 rata,β2 adrenoceptor-mediated relaxation of airway smooth muscle pre-contracted with 5-HT was inhibited,and there were no obvious difference in relaxation compared with normal E3 rats.Contraction of lobar bronchi mediated by 5-HT and sarafotoxin 6c was more potent than in the trachea or main bronchi.Airway contractions mediated by the endothelin (ET)A receptor,ETB receptor and M3 muscarinic receptor were augmented,and the augmented contraction was most obvious in lobar bronchi.The order of efficacy of contraction for lobar bronchi induced by agonists was ET-1,sarafotoxin 6c>ACh>5-HT.OX8 (an antibody against CD8+ T cells) strongly shifted and 0X35 (an antibody against CD4+ T cells) modestly shifted isoprenaline-induced concentration-relaxation curves in a nonparallel fashion to the left with an increased Rmax in asthmatic rats and sarafotoxin 6c-induced concentration-contractile curves to the right with a decreased Emax.Conclusion:The inhibition of airway relaxation and the augmentation of contraction mediated by receptors contribute to airway hyperresponsiveness and involve CD8+ and CD4+ T cells.

  1. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression. (United States)

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-Il; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y L; Choi, Hueng-Sik


    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism.

  2. Transmembrane α-Helix 2 and 7 Are Important for Small Molecule-Mediated Activation of the GLP-1 Receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye; Møller Knudsen, Sanne; Schjellerup Wulff, Birgitte;


    , the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R...

  3. Hippocampus NMDA receptors selectively mediate latent extinction of place learning. (United States)

    Goodman, Jarid; Gabriele, Amanda; Packard, Mark G


    Extinction of maze learning may be achieved with or without the animal performing the previously acquired response. In typical "response extinction," animals are given the opportunity to make the previously acquired approach response toward the goal location of the maze without reinforcement. In "latent extinction," animals are not given the opportunity to make the previously acquired response and instead are confined to the previous goal location without reinforcement. Previous evidence indicates that the effectiveness of these protocols may depend on the type of memory being extinguished. Thus, one aim of the present study was to further examine the effectiveness of response and latent extinction protocols across dorsolateral striatum (DLS)-dependent response learning and hippocampus-dependent place learning tasks. In addition, previous neural inactivation experiments indicate a selective role for the hippocampus in latent extinction, but have not investigated the precise neurotransmitter mechanisms involved. Thus, the present study also examined whether latent extinction of place learning might depend on NMDA receptor activity in the hippocampus. In experiment 1, adult male Long-Evans rats were trained in a response learning task in a water plus-maze, in which animals were reinforced to make a consistent body-turn response to reach an invisible escape platform. Results indicated that response extinction, but not latent extinction, was effective at extinguishing memory in the response learning task. In experiment 2, rats were trained in a place learning task, in which animals were reinforced to approach a consistent spatial location containing the hidden escape platform. In experiment 2, animals also received intra-hippocampal infusions of the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid (AP5; 5.0 or 7.5 ug/0.5 µg) or saline vehicle immediately before response or latent extinction training. Results indicated that both extinction protocols were

  4. Defective renal dopamine D1 receptor function contributes to hyperinsulinemia-mediated hypertension. (United States)

    Ahmad Banday, Anees; Lokhandwala, Mustafa F


    Hyperinsulinemia is reported to play a role in hypertension, as abnormalities in blood pressure regulation and sodium handling exist in diabetes mellitus. Kidney dopamine promotes sodium excretion via the activation of renal D1 receptors. Because there is a close relationship between renal D1 receptor function and sodium excretion, it is hypothesized that a defect in this mechanism may contribute to decreased sodium excretion and hypertension during hyperinsulinemia. Renal D1 receptor function was studied in insulin-induced hypertension in male Sprague Dawley rats. Insulin pellets were implanted subcutaneously for controlled insulin release for three weeks; sham rats served as a control. Compared to control rats, insulin pellets increased plasma insulin levels by eight fold and decreased blood glucose by 40%. Insulin also caused a 22 mmHg increase in mean arterial blood pressure compared to control animals. The intravenous infusion of SKF-38393, a D1 receptor agonist, increased sodium excretion in control rats, but SKF-38393 failed to produce natriuresis in hyperinsulinemic animals. Renal proximal tubules from hyperinsulinemic rats had a reduced D1 receptor number, defective receptor-G protein coupling, and blunted SKF-38393 induced Na, K-ATPase inhibition. Insulin seems to reduce D1 receptor expression and coupling to the G-protein, leading to a reduced D1 receptor-mediated Na, K-ATPase inhibition, and a diminished natriuretic response to SKF-38393. These phenomena could account for sodium retention and hypertension associated with hyperinsulinemia.

  5. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes (United States)

    Ruiz, A; Matute, C; Alberdi, E


    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  6. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. (United States)

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu


    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  7. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission. (United States)

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia


    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  8. The use of occupation isoboles for analysis of a response mediated by two receptors: M2 and M3 muscarinic receptor subtype-induced mouse stomach contractions. (United States)

    Braverman, Alan S; Tallarida, Ronald J; Ruggieri, Michael R


    Smooth muscle contains multiple muscarinic receptor subtypes, including M2 and M3. M2 receptors outnumber M3 receptors. Based on the potency of subtype selective anticholinergics, contraction is mediated by the M3 subtype. However, results from knockout (KO) mice show that the M2 receptor mediates approximately 45% of the contractile response produced by the M3 receptor. The traditional theory of one receptor mediating a response does not allow assessment of interactions between receptors when more than one receptor participates in a response. Our study was performed using a novel analysis method based on dual receptor occupancy to determine how M2 and M3 receptor subtypes interact to mediate contraction in mouse stomach. Cumulative carbachol concentration contractile responses were determined for wild-type, M2-KO, and M3-KO stomach body smooth muscle. Using affinity constants for carbachol at M2 and M3 cholinergic receptors, the concentration values were converted to fractional receptor occupation. The resulting occupation-effect relations showed maximum effects for the M2 and M3 subtypes, respectively. These occupation-effect relations allow determination of the additive (expected) isobole based on this dual occupancy, thereby providing a curve (mathematically derived) for comparison against the experimentally derived value in wild type. The actual values determined experimentally in the wild type were not statistically significantly different from that predicted by the isobole. This confirms that the interaction between these mutually occupied receptors is additive. The new method of analysis also expands the traditional Schild theory that was based on a single receptor type to which the agonist and antagonist bind.

  9. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius


    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......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...

  10. Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion.

    Directory of Open Access Journals (Sweden)

    Alex de Voux

    Full Text Available The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·⁴⁹(¹²⁵ and Arg⁶·³²(²²⁵ residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·⁵⁶(⁸², in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr²·⁵⁶(⁸² mutants were fully stabilized in active conformations. The Thr²·⁵⁶(⁸²Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·⁵⁶(⁸²Lys mutation with an Arg⁶·³²(²²⁵Gln mutation partially reversed the decrease in expression. Mutants with Thr²·⁵⁶(⁸²Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·⁶⁵(⁸²Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·⁶⁵(⁸²Lys and Thr²·⁶⁵(⁸²Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82 stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated

  11. Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion. (United States)

    de Voux, Alex; Chan, Mei-Chi; Folefoc, Asongna T; Madziva, Michael T; Flanagan, Colleen A


    The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·⁴⁹(¹²⁵) and Arg⁶·³²(²²⁵) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·⁵⁶(⁸²), in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr²·⁵⁶(⁸²) mutants were fully stabilized in active conformations. The Thr²·⁵⁶(⁸²)Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·⁵⁶(⁸²)Lys mutation with an Arg⁶·³²(²²⁵)Gln mutation partially reversed the decrease in expression. Mutants with Thr²·⁵⁶(⁸²)Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·⁶⁵(⁸²)Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·⁶⁵(⁸²)Lys and Thr²·⁶⁵(⁸²)Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations

  12. Potentiation of NMDA receptor-mediated transmission in striatal cholinergic interneurons

    Directory of Open Access Journals (Sweden)

    Manfred eOswald


    Full Text Available Pauses in the tonic firing of striatal cholinergic interneurons (CINs emerge during reward-related learning in response to conditioning of a neutral cue. We have previously reported that augmenting the postsynaptic response to cortical afferents in CINs is coupled to the emergence of a cell-intrinsic afterhyperpolarisation (AHP underlying pauses in tonic activity. Here we investigated in a bihemispheric rat-brain slice preparation the mechanisms of synaptic plasticity of excitatory afferents to CINs and the association with changes in the AHP. We found that high frequency stimulation (HFS of commissural corticostriatal afferents from the contralateral hemisphere induced a robust long-term depression (LTD of postsynaptic potentials (PSP in CINs. Depression of the PSP of smaller magnitude and duration was observed in response to HFS of the ipsilateral white matter or cerebral cortex. In Mg2+-free solution HFS induced NMDA receptor-dependent potentiation of the PSP, evident in both the maximal slope and amplitude of the PSP. The increase in maximal slope corroborates previous findings, and was blocked by antagonism of either D1-like dopamine receptors with SCH23390 or D2-like dopamine receptors with sulpiride during HFS in Mg2+-free solution. Potentiation of the slower PSP amplitude component was due to augmentation of the NMDA receptor-mediated potential as this was completely reversed on subsequent application of the NMDA receptor antagonist AP5. HFS similarly potentiated NMDA receptor currents isolated by blockade of AMPA/kainate receptors with CNQX. The plasticity-induced increase in the slow PSP component was directly associated with an increase in the subsequent AHP. Thus plasticity of cortical afferent synapses is ideally suited to influence the cue-induced firing dynamics of CINs, particularly through potentiation of NMDA receptor-mediated synaptic transmission.

  13. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Siti H Sheikh Abdul Kadir

    Full Text Available BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC, which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM. Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart. METHODS AND RESULTS: Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2 receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2 receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters. CONCLUSION: We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2 receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

  14. Muscarinic receptor-mediated calcium changes in a rat model of facial nerve nucleus injury

    Institute of Scientific and Technical Information of China (English)

    Dawei Sun; Huamin Liu; Fugao Zhu; Yanqing Wang; Junfeng Wen; Rui Zhou; Yanjun Wang; Banghua Liu


    The muscarinic receptor modulates intracellular free calcium ion levels in the facial nerve nucleus via different channels.In the present study,muscarinic receptor-mediated free calcium ions levels were detected by confocal laser microscopy in the facial nerve nucleus following facial nerve injury in rats.There was no significant difference in muscarinic receptor expression at the affected facial nerve nucleus compared with expression prior to injury,but muscarinic receptor-mediated free calcium ion levels increased in the affected side following facial nerve injury(P < 0.01).At day 30after facial nerve injury,50 μmol/L muscarinic-mediated free calcium ion levels were significantly inhibited at the affected facial nerve nucleus in calcium-free artificial cerebrospinal fluid,and the change range was 82% of artificial cerebrospinal fluid(P < 0.05).These results suggest that increased free calcium ion concentrations are achieved by intracellular calcium ion release,and that the transmembrane flow of calcium ions is also involved in this process.

  15. Sphingosine 1-phosphate receptors are essential mediators of eyelid closure during embryonic development. (United States)

    Herr, Deron R; Lee, Chang-Wook; Wang, Wei; Ware, Adam; Rivera, Richard; Chun, Jerold


    The fetal development of the mammalian eyelid involves the expansion of the epithelium over the developing cornea, fusion into a continuous sheet covering the eye, and a splitting event several weeks later that results in the formation of the upper and lower eyelids. Recent studies have revealed a significant number of molecular signaling components that are essential mediators of eyelid development. Receptor-mediated sphingosine 1-phosphate (S1P) signaling is known to influence diverse biological processes, but its involvement in eyelid development has not been reported. Here, we show that two S1P receptors, S1P2 and S1P3, are collectively essential mediators of eyelid closure during murine development. Homozygous deletion of the gene encoding either receptor has no apparent effect on eyelid development, but double-null embryos are born with an "eyes open at birth" defect due to a delay in epithelial sheet extension. Both receptors are expressed in the advancing epithelial sheet during the critical period of extension. Fibroblasts derived from double-null embryos have a deficient response to epidermal growth factor, suggesting that S1P2 and S1P3 modulate this essential signaling pathway during eyelid closure.

  16. Expression of folate receptors in nasopharyngeal and laryngeal carcinoma and folate receptor-mediated endocytosis by molecular targeted nanomedicine

    Directory of Open Access Journals (Sweden)

    Xie M


    Full Text Available M Xie, H Zhang, Y Xu, T Liu, S Chen, J Wang, T ZhangDepartment of Otorhinolaryngology Head and Neck Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of ChinaAbstract: Immunohistochemistry and an immunofluorescence technique was used to detect folate receptor expression in tissue samples and cell lines of head and neck squamous carcinoma, including 20 tissue samples of nasopharyngeal carcinoma, 16 tissue samples of laryngeal carcinoma, and HNE-1, HNE-2, CNE-1, CNE-2, SUNE-1, 5-8F, and Hep-2 cell lines. Iron staining, electron microscopy, and magnetic resonance imaging were used to observe endocytosis of folate-conjugated cisplatin-loaded magnetic nanoparticles (CDDP-FA-ASA-MNP in cultured cells and transplanted tumors. As shown by immunohistochemistry, 83.3% (30/36 of the head and neck squamous carcinomas expressed the folate receptor versus none in the control group (0/24. Only the HNE-1 and Hep-2 cell lines expressed the folate receptor, and the other five cell lines did not. Endocytosis of CDDP-FA-ASA-MNP was seen in HNE-1 and Hep-2 cells by iron staining and electron microscopy. A similar result was seen in transplanted tumors in nude mice. Magnetic resonance imaging showed low signal intensity of HNE-1 cells and HNE-1 transplanted tumors on T2-weighted images after uptake of CDDP-FA-ASA-MNP, and this was not seen in CNE-2 transplanted tumors. In conclusion, head and neck squamous carcinoma cell strongly expressed the folate receptor, while normal tissue did not. The folate receptor can mediate endocytosis of folate-conjugated anticancer nanomedicines, and lays the foundation for molecular targeted treatment of cancer.Keywords: nasopharyngeal carcinoma, laryngeal carcinoma, folate receptor, molecular targeting, cisplatin, nanomedicine

  17. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor. (United States)

    Murphy, Jane E; Roosterman, Dirk; Cottrell, Graeme S; Padilla, Benjamin E; Feld, Micha; Brand, Eva; Cedron, Wendy J; Bunnett, Nigel W; Steinhoff, Martin


    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK(1)R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK(1)R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca(2+) signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK(1)R. SP induced association of β-arrestin1 and PP2A with noninternalized NK(1)R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK(1)R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK(1)R with PP2A. Resensitization of NK(1)R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK(1)R and mediates resensitization. PP2A interaction with NK(1)R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK(1)R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs.

  18. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH


    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  19. Kainate receptors mediate signaling in both transient and sustained OFF bipolar cell pathways in mouse retina. (United States)

    Borghuis, Bart G; Looger, Loren L; Tomita, Susumu; Demb, Jonathan B


    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.

  20. Nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system

    Institute of Scientific and Technical Information of China (English)

    Jian-xin SHEN; Jerrel L YAKEL


    Based on the composition of the five subunits forming functional neuronal nicotinic acetylcholine receptors (nAChRs), they are grouped into either heteromeric (comprising both α and β subunits) or homomeric (comprising only α subunits) recep-tors. The nAChRs are known to be differentially permeable to calcium ions, with the α7 nAChR subtype having one of the highest permeabilities to calcium. Calcium influx through nAChRs, particularly through the α-bungarotoxin-sensitive α7-containing nAChRs, is a very efficient way to raise cytoplasmic calcium levels. The activation of nAChRs can mediate three types of cytoplasmic calcium signals: (1) direct calcium influx through the nAChRs, (2) indirect calcium influx through voltage-dependent calcium channels (VDCCs) which are activated by the nAChR-mediated depolarization, and (3) calcium-induced calcium release (CICR) (triggered by the first two sources) from the endoplasmic reticulum (ER) through the ryanodine receptors and inositol (1,4,5)-triphosphate receptors (IP3Rs). Downstream signaling events mediated by nAChR-mediated calcium responses can be grouped into instantaneous effects (such as neurotransmitter release, which can occur in milliseconds after nAChR activation), short-term effects (such as the recovery of nAChR desensitization through cellular signaling cascades), and long-term effects (such as neuroprotection via gene expression). In addition, nAChR activity can be regulated by cytoplasmic calcium levels, suggesting a complex reciprocal relationship. Further advances in imaging techniques, animal models, and more potent and subtype-selective ligands for neuronal nAChRs would help in understand-ing the neuronal nAChR-mediated calcium signaling, and lead to the development of improved therapeutic treatments.

  1. Brain delta2 opioid receptors mediate SNC-80-evoked hypothermia in rats. (United States)

    Rawls, Scott Manning; Hewson, Jennifer Marie; Inan, Saadet; Cowan, Alan


    Despite insights into an increasingly significant role for delta opioid receptors in thermoregulation, it is unclear whether delta receptors located in the brain or periphery play the more critical role in body temperature regulation. Moreover, it is not entirely clear which delta receptor phenotype, delta1 or delta2, mediates the hypothermic actions of delta agonists. Because SNC-80 distributes into central and peripheral compartments and produces rapid hypothermia following systemic injection, the nonpeptide delta agonist is particularly useful in discriminating the site of action of delta receptor-mediated hypothermia. To determine the locus and phenotype of delta receptor which mediates SNC-80-induced hypothermia, we injected SNC-80 and phenotype selective delta antagonists to male Sprague-Dawley rats. SNC-80 (10-50 mg/kg, im) evoked hypothermia that peaked 30 min post-injection. Naltrexone (5 mg/kg, sc), an opioid antagonist, or naltrindole (5 mg/kg, sc), a delta antagonist, blocked the hypothermic response to SNC-80 (35 mg/kg, im). The hypothermia caused by SNC-80 (35 mg/kg, im) was blocked by a delta2 antagonist, naltriben (2.5 mg/kg, sc), but was not affected by BNTX (5 and 10 mg/kg, sc), a delta1 antagonist. The administration of naltriben (10 microg/rat, icv) 30 min before SNC-80 (35 mg/kg, im) prevented SNC-80-evoked hypothermia. In contrast, methylnaltrexone (5 mg/kg, sc), a peripherally restricted opioid antagonist, did not affect the hypothermia caused by SNC-80. The present data demonstrate that selective activation of brain delta2 receptors is a major mechanism of SNC-80-evoked hypothermia in rats.

  2. AMPA receptor mediated excitotoxicity in neocortical neurons is developmentally regulated and dependent upon receptor desensitization

    DEFF Research Database (Denmark)

    Jensen, J B; Schousboe, A; Pickering, D S;


    was blocked was seen as early as 5 DIV since 10 microM MK-801 did not completely block the response whereas 10 microM NBQX did. The 2,3-benzodiazepine GYKI compounds, which have been reported to be selective non-competitive AMPA receptor antagonists, were here observed to block the AMPA toxicity...

  3. Role of IL-33 and Its Receptor in T Cell-Mediated Autoimmune Diseases

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    Qing Zhao


    Full Text Available Interleukin-33 (IL-33 is a new cytokine of interleukin-1 family, whose specific receptor is ST2. IL-33 exerts its functions via its target cells and plays different roles in diseases. ST2 deletion and exclusion of IL-33/ST2 axis are accompanied by enhanced susceptibility to dominantly T cell-mediated organ-specific autoimmune diseases. It has been reported that IL-33/ST2 pathway plays a key role in host defense and immune regulation in inflammatory and infectious diseases. This review focuses on new findings in the roles of IL-33 and ST2 in several kinds of T cell-mediated autoimmune diseases.

  4. Interaction of medullary P2 and glutamate receptors mediates the vasodilation in the hindlimb of rat. (United States)

    Korim, Willian Seiji; Ferreira-Neto, Marcos L; Pedrino, Gustavo R; Pilowsky, Paul M; Cravo, Sergio L


    In the nucleus tractus solitarii (NTS) of rats, blockade of extracellular ATP breakdown to adenosine reduces arterial blood pressure (AP) increases that follow stimulation of the hypothalamic defense area (HDA). The effects of ATP on NTS P2 receptors, during stimulation of the HDA, are still unclear. The aim of this study was to determine whether activation of P2 receptors in the NTS mediates cardiovascular responses to HDA stimulation. Further investigation was taken to establish if changes in hindlimb vascular conductance (HVC) elicited by electrical stimulation of the HDA, or activation of P2 receptors in the NTS, are relayed in the rostral ventrolateral medulla (RVLM); and if those responses depend on glutamate release by ATP acting on presynaptic terminals. In anesthetized and paralyzed rats, electrical stimulation of the HDA increased AP and HVC. Blockade of P2 or glutamate receptors in the NTS, with bilateral microinjections of suramin (10 mM) or kynurenate (50 mM) reduced only the evoked increase in HVC by 75 % or more. Similar results were obtained with the blockade combining both antagonists. Blockade of P2 and glutamate receptors in the RVLM also reduced the increases in HVC to stimulation of the HDA by up to 75 %. Bilateral microinjections of kynurenate in the RVLM abolished changes in AP and HVC to injections of the P2 receptor agonist α,β-methylene ATP (20 mM) into the NTS. The findings suggest that HDA-NTS-RVLM pathways in control of HVC are mediated by activation of P2 and glutamate receptors in the brainstem in alerting-defense reactions.

  5. Vascular endothelin ET(B) receptor-mediated contraction requires phosphorylation of ERK1/2 proteins

    DEFF Research Database (Denmark)

    Luo, Guogang; Jamali, Roya; Cao, Yong-Xiao;


    RNA and protein expressions. The endothelin ET(B) receptor-mediated contraction was associated with increase in phosphorylation of extracellular regulation kinase 1 and 2 (ERK1/2) proteins and elevated levels of intracellular calcium. The elevation curve of intracellular calcium consisted of two phases: one rapid...... and one sustained. Inhibition of ERK1/2 phosphorylation by SB386023 or blockage of calcium channels by nifedipine significantly reduced the endothelin ET(B) receptor-mediated contraction (P..., phosphorylation of ERK1/2 proteins and elevation of intracellular calcium level are required for endothelin ET(B) receptor-mediated contraction in rat mesenteric artery....

  6. AT1 receptor in rostral ventrolateral medulla mediating blunted baroreceptor reflex in spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    Xin-yaGAO; FengZHANG; YingHAN; Han-junWANG; YingZHANG; RuiGUO; Guo-qingZHU


    AIM: To determine the role of AT1 receptor in the rostral ventrolateral medulla (RVLM) in mediating the blunted baroreceptor reflex in spontaneously hypertensive rats (SHR). METHODS: Intravenous injections of graded doses of phenylephrine (1, 5, 10, 20, and 40μg/kg) increased the blood pressure to elicit the baroreceptor reflex in both SHR and normotensive Wistar rats anesthetized with urethane. The baroreceptor reflex sensitivity (BRS) was determined before and after microinjection of Ang II, losartan, or AT1 receptor antisense oligodeoxynucleotides into the RVLM. AT1 receptor protein level in the RVLM was measured by Western blotting. RESULTS: The BRS was significantly decreased in SHR compared with normal rats. Bilateral microinjection of AT~ receptor antagonist losartan (250 nmol/h) into the RVLM partly reversed the blunted BRS in SHR, but had no significant effect on the BRS in normal rats. Ang II (1.5 nmol/h) significantly inhibited the BRS in normal rats, which was completely abolished by pretreatment with losartan. However, no significant change in the BRS was observed after microinjection of Ang Ⅱ into the RVLM in SHR. Bilateral microinjection of AT1 receptor antisense oligodeoxynucleotides (ASODN) into the RVLM partially recovered the blunted BRS in SHR after 3 h, but no significant change in the BRS was observed in normal rats. The AT1 receptor protein level significantly decreased after administration of ASODN. CONCLUSION: Blockage of AT1 receptor or inhibition of AT1 receptor protein synthesis in the RVLM enhanced the BRS in SHR, suggesting that the enhanced activities of AT1 receptor in the RVLM contribute to the blunted BRS in SHR.

  7. AT1 receptor in rostral ventrolateral medulla mediating blunted baroreceptor reflex in spontaneously hypertensive rats

    Institute of Scientific and Technical Information of China (English)

    Xin-ya GAO; Feng ZHANG; Ying HAN; Han-jun WANG; Ying ZHANG; Rui GUO; Guo-qing ZHU


    AIM: To determine the role of AT1 receptor in the rostral ventrolateral medulla (RVLM) in mediating the blunted baroreceptor reflex in spontaneously hypertensive rats (SHR). METHODS: Intravenous injections of graded doses of phenylephrine (1, 5, 10, 20, and 40 μg/kg) increased the blood pressure to elicit the baroreceptor reflex in both SHR and normotensive Wistar rats anesthetized with urethane. The baroreceptor reflex sensitivity (BRS) was determined before and after microinjection of Ang Ⅱ, losartan, or AT1 receptor antisense oligodeoxynucleotides into the RVLM. AT1 receptor protein level in the RVLM was measured by Western blotting. RESULTS: The BRS was significantly decreased in SHR compared with normal rats. Bilateral microinjection of AT1 receptor antagonist losartan (250 nmol/h) into the RVLM partly reversed the blunted BRS in SHR, but had no significant effect on the BRS in normal rats. Ang Ⅱ (1.5 nmol/h) significantly inhibited the BRS in normal rats, which was completely abolished by pretreatment with losartan. However, no significant change in the BRS was observed after microinjection of Ang Ⅱ into the RVLM in SHR. Bilateral microinjection of AT1 receptor antisense oligodeoxynucleotides(ASODN) into the RVLM partially recovered the blunted BRS in SHR after 3 h, but no significant change in the BRS was observed in normal rats. The AT1 receptor protein level significantly decreased after administration of ASODN.CONCLUSION: Blockage of AT1 receptor or inhibition of AT1 receptor protein synthesis in the RVLM enhanced the BRS in SHR, suggesting that the enhanced activities of AT1 receptor in the RVLM contribute to the blunted BRS in SHR.

  8. Activation of cortical 5-HT3 receptor-expressing interneurons induces NO mediated vasodilatations and NPY mediated vasoconstrictions

    Directory of Open Access Journals (Sweden)

    Quentin ePerrenoud


    Full Text Available GABAergic interneurons are local integrators of cortical activity that have been reported to be involved in the control of cerebral blood flow through their ability to produce vasoactive molecules and their rich innervation of neighboring blood vessels. They form a highly diverse population among which the serotonin 5-hydroxytryptamine 3A receptor (5-HT3A-expressing interneurons share a common developmental origin, in addition to the responsiveness to serotonergic ascending pathway. We have recently shown that these neurons regroup two distinct subpopulations within the somatosensory cortex: Neuropeptide Y (NPY-expressing interneurons, displaying morphological properties similar to those of neurogliaform cells, and Vasoactive Intestinal Peptide (VIP-expressing bipolar/bitufted interneurons. The aim of the present study was to determine the role of these neuronal populations in the control of vascular tone by monitoring blood vessels diameter changes, using infrared videomicroscopy in mouse neocortical slices. Bath applications of 1-(3-Chlorophenylbiguanide hydrochloride (mCPBG, a 5-HT3R agonist, induced both constrictions (30% and dilations (70% of penetrating arterioles within supragranular layers. All vasoconstrictions were abolished in the presence of the NPY receptor antagonist (BIBP 3226, suggesting that they were elicited by NPY release. Vasodilations persisted in the presence of the VIP receptor antagonist VPAC1 (PG-97-269, whereas they were blocked in the presence of the neuronal Nitric Oxide (NO Synthase (nNOS inhibitor, L-NNA. Altogether, these results strongly suggest that activation of neocortical 5-HT3A-expressing interneurons by serotoninergic input could induces NO mediated vasodilatations and NPY mediated vasoconstrictions.

  9. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain. (United States)

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E


    , 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.

  10. Enterotoxin preconditioning restores calcium-sensing receptor-mediated cytostasis in colon cancer cells


    Pitari, Giovanni M.; Lin, Jieru E.; Shah, Fawad J.; Lubbe, Wilhelm J.; Zuzga, David S.; Li, Peng; Schulz, Stephanie; Waldman, Scott A.


    Guanylyl cyclase C (GCC), the receptor for diarrheagenic bacterial heat-stable enterotoxins (STs), inhibits colorectal cancer cell proliferation by co-opting Ca2+ as the intracellular messenger. Similarly, extracellular Ca2+ (Ca2+o) opposes proliferation and induces terminal differentiation in intestinal epithelial cells. In that context, human colon cancer cells develop a phenotype characterized by insensitivity to cytostasis imposed by Ca2+o. Here, preconditioning with ST, mediated by GCC s...

  11. Metabotropic Glutamate Receptor-Mediated Long-Term Depression: Molecular Mechanisms


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


    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 ...

  12. THIP, a hypnotic and antinociceptive drug, enhances a tonic GABAA receptor mediated conductance in mouse neocortex

    DEFF Research Database (Denmark)

    Drasbek, Kim Ryun; Jensen, Kimmo


    its cellular actions in the neocortex are uncertain, we studied the effects of THIP on neurons in slices of frontoparietal neocortex of 13- to 19-day-old (P13-19) mice. Using whole-cell patch-clamp recordings, we found that the clinically relevant THIP concentration of 1 μM induced a robust tonic GABA...... suggest that THIP activates an extrasynaptic GABA(A) receptor-mediated conductance in the neocortex, which may alter the cortical network activity....

  13. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress. (United States)

    Evanson, Nathan K; Herman, James P


    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  14. New Insights into VacA Intoxication Mediated through Its Cell Surface Receptors

    Directory of Open Access Journals (Sweden)

    Kinnosuke Yahiro


    Full Text Available Helicobacter pylori (H. pylori, a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPβ, and LRP1 that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage.

  15. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis. (United States)

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


    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.

  16. Slit2 involvement in glioma cell migration is mediated by Robo1 receptor. (United States)

    Mertsch, Sonja; Schmitz, Nicole; Jeibmann, Astrid; Geng, Jian-Guo; Paulus, Werner; Senner, Volker


    Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRT-PCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels. Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2 receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

  17. AB318. SPR-45 Decentralization reduces nicotinic receptor-mediated canine bladder contractions in vitro (United States)

    Salvadeo, Danielle M.; Frara, Nagat; Braverman, Alan S.; Barbe, Mary F.; Ruggieri, Michael R.


    had no significant inhibitory effect on DMPP, epibatidine or nicotine-induced contraction in any group. Conclusions Nicotinic receptors mediate contraction in sham, reinnervated and decentralized bladders. This nicotinic receptor-mediated contraction is decreased after decentralization. TTX does not block nicotinic receptor-mediated contractions, indicating that action potentials are not required to induce contraction. In sham-operated dog bladders, the nicotine-induced contraction is blocked by ATR, suggesting that these nicotinic receptors are located on cholinergic nerve terminals and induce the release of acetylcholine, which activates muscarinic receptors on the smooth muscle. Funding Source(s) NIH-NINDS NS070267

  18. MicroRNA-219 modulates NMDA receptor-mediated neurobehavioral dysfunction

    DEFF Research Database (Denmark)

    Kocerha, Jannet; Faghihi, Mohammad Ali; Lopez-Toledano, Miguel A;


    N-methyl-D-aspartate (NMDA) glutamate receptors are regulators of fast neurotransmission and synaptic plasticity in the brain. Disruption of NMDA-mediated glutamate signaling has been linked to behavioral deficits displayed in psychiatric disorders such as schizophrenia. Recently, noncoding RNA...... significantly modulated behavioral responses associated with disrupted NMDA receptor transmission. Furthermore, pretreatment with the antipsychotic drugs haloperidol and clozapine prevented dizocilpine-induced effects on miR-219. Taken together, these data support an integral role for miR-219 in the expression...... molecules such as microRNAs (miRNAs) have emerged as critical regulators of neuronal functions. Here we show that pharmacological (dizocilpine) or genetic (NR1 hypomorphism) disruption of NMDA receptor signaling reduces levels of a brain-specific miRNA, miR-219, in the prefrontal cortex (PFC) of mice...

  19. Inflammatory Mediators and Insulin Resistance in Obesity: Role of Nuclear Receptor Signaling in Macrophages

    Directory of Open Access Journals (Sweden)

    Lucía Fuentes


    Full Text Available Visceral obesity is coupled to a general low-grade chronic inflammatory state characterized by macrophage activation and inflammatory cytokine production, leading to insulin resistance (IR. The balance between proinflammatory M1 and antiinflammatory M2 macrophage phenotypes within visceral adipose tissue appears to be crucially involved in the development of obesity-associated IR and consequent metabolic abnormalities. The ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs have recently been implicated in the determination of the M1/M2 phenotype. Liver X receptors (LXRs, which form another subgroup of the nuclear receptor superfamily, are also important regulators of proinflammatory cytokine production in macrophages. Disregulation of macrophage-mediated inflammation by PPARs and LXRs therefore underlies the development of IR. This review summarizes the role of PPAR and LXR signaling in macrophages and current knowledge about the impact of these actions in the manifestation of IR and obesity comorbidities such as liver steatosis and diabetic osteopenia.

  20. IL-6 Overexpression in ERG-Positive Prostate Cancer Is Mediated by Prostaglandin Receptor EP2. (United States)

    Merz, Constanze; von Mässenhausen, Anne; Queisser, Angela; Vogel, Wenzel; Andrén, Ove; Kirfel, Jutta; Duensing, Stefan; Perner, Sven; Nowak, Michael


    Prostate cancer is the most diagnosed cancer in men and multiple risk factors and genetic alterations have been described. The TMPRSS2-ERG fusion event and the overexpression of the transcription factor ERG are present in approximately 50% of all prostate cancer patients, however, the clinical outcome is still controversial. Prostate tumors produce various soluble factors, including the pleiotropic cytokine IL-6, regulating cellular processes such as proliferation and metastatic segregation. Here, we used prostatectomy samples in a tissue microarray format and analyzed the co-expression and the clinicopathologic data of ERG and IL-6 using immunohistochemical double staining and correlated the read-out with clinicopathologic data. Expression of ERG and IL-6 correlated strongly in prostate tissue samples. Forced expression of ERG in prostate tumor cell lines resulted in significantly increased secretion of IL-6, whereas the down-regulation of ERG decreased IL-6 secretion. By dissecting the underlying mechanism in prostate tumor cell lines we show the ERG-mediated up-regulation of the prostanoid receptors EP2 and EP3. The prostanoid receptor EP2 was overexpressed in human prostate cancer tissue. Furthermore, the proliferation rate and IL-6 secretion in DU145 cells was reduced after treatment with EP2-receptor antagonist. Collectively, our study shows that the expression of ERG in prostate cancer is linked to the expression of IL-6 mediated by the prostanoid receptor EP2.

  1. Evidence that the anorexia induced by lipopolysaccharide is mediated by the 5-HT2C receptor. (United States)

    von Meyenburg, Claudia; Langhans, Wolfgang; Hrupka, Brian J


    Rats consistently reduce their food intake following injections of bacterial lipopolysaccharides (LPS). Because inhibition of serotonergic (5-HT) activity by 8-OH-DPAT (5-HT(1A) activation) attenuates LPS-induced anorexia, we conducted a series of studies to examine whether other 5-HT-receptors are involved in the mediation of peripheral LPS-induced anorexia. In all experiments, rats were injected with LPS (100 microg/kg body weight [BW] ip) at lights out (hour 0). Antagonists were administered peripherally at hour 4, shortly after the onset of anorexia, which presumably follows the enhanced cytokine production after LPS. Food intake was then recorded during the subsequent 2 h or longer. 5-HT receptor antagonists cyanopindolol and SB 224289 (5-HT(1B)), ketanserin (5-HT(2A)), RS-102221 (5-HT(2C)), and metoclopramide (5-HT(3)) failed to attenuate LPS-induced anorexia. In contrast, both ritanserin (5-HT(2A/C)-receptor antagonist) (0.5 mg/kg BW) and SB 242084 (5-HT(2C)) (0.3 mg/kg BW) attenuated LPS-induced anorexia at doses that did not alter food intake in non-LPS-treated rats (all Panorexia following peripheral LPS administration is mediated through an enhanced 5-HT-ergic activity and the 5-HT(2C) receptor.

  2. Protein kinase Czeta mediates micro-opioid receptor-induced cross-desensitization of chemokine receptor CCR5. (United States)

    Song, Changcheng; Rahim, Rahil T; Davey, Penelope C; Bednar, Filip; Bardi, Giuseppe; Zhang, Lily; Zhang, Ning; Oppenheim, Joost J; Rogers, Thomas J


    We have previously shown that the μ-opioid receptor (MOR) is capable of mediating cross-desensitization of several chemokine receptors including CCR5, but the biochemical mechanism of this process has not been fully elucidated. We have carried out a series of functional and biochemical studies and found that the mechanism of MOR-induced cross-desensitization of CCR5 involves the activation of PKCζ. Inhibition of PKCζ by its pseudosubstrate inhibitor, or its siRNA, or dominant negative mutants suppresses the cross-desensitization of CCR5. Our results further indicate that the activation of PKCζ is mediated through a pathway involving phosphoinositol-dependent kinase-1 (PDK1). In addition, activation of MOR elevates the phosphorylation level and kinase activity of PKCζ. The phosphorylation of PKCζ can be suppressed by a dominant negative mutant of PDK1. We observed that following MOR activation, the interaction between PKCζ and PDK1 is immediately increased based on the analysis of fluorescent resonance energy transfer in cells with the expression of PKCζ-YFP and PDK1-CFP. In addition, cells expressing PKCζ kinase motif mutants (Lys-281, Thr-410, Thr-560) fail to exhibit full MOR-induced desensitization of CCR5 activity. Taken together, we propose that upon DAMGO treatment, MOR activates PKCζ through a PDK1-dependent signaling pathway to induce CCR5 phosphorylation and desensitization. Because CCR5 is a highly proinflammatory receptor, and a critical coreceptor for HIV-1, these results may provide a novel approach for the development of specific therapeutic agents to treat patients with certain inflammatory diseases or AIDS.

  3. GRK2 protein-mediated transphosphorylation contributes to loss of function of μ-opioid receptors induced by neuropeptide FF (NPFF2) receptors. (United States)

    Moulédous, Lionel; Froment, Carine; Dauvillier, Stéphanie; Burlet-Schiltz, Odile; Zajac, Jean-Marie; Mollereau, Catherine


    Neuropeptide FF (NPFF) interacts with specific receptors to modulate opioid functions in the central nervous system. On dissociated neurons and neuroblastoma cells (SH-SY5Y) transfected with NPFF receptors, NPFF acts as a functional antagonist of μ-opioid (MOP) receptors by attenuating the opioid-induced inhibition of calcium conductance. In the SH-SY5Y model, MOP and NPFF(2) receptors have been shown to heteromerize. To understand the molecular mechanism involved in the anti-opioid activity of NPFF, we have investigated the phosphorylation status of the MOP receptor using phospho-specific antibody and mass spectrometry. Similarly to direct opioid receptor stimulation, activation of the NPFF(2) receptor by [D-Tyr-1-(NMe)Phe-3]NPFF (1DMe), an analog of NPFF, induced the phosphorylation of Ser-377 of the human MOP receptor. This heterologous phosphorylation was unaffected by inhibition of second messenger-dependent kinases and, contrarily to homologous phosphorylation, was prevented by inactivation of G(i/o) proteins by pertussis toxin. Using siRNA knockdown we could demonstrate that 1DMe-induced Ser-377 cross-phosphorylation and MOP receptor loss of function were mediated by the G protein receptor kinase GRK2. In addition, mass spectrometric analysis revealed that the phosphorylation pattern of MOP receptors was qualitatively similar after treatment with the MOP agonist Tyr-D-Ala-Gly (NMe)-Phe-Gly-ol (DAMGO) or after treatment with the NPFF agonist 1DMe, but the level of multiple phosphorylation was more intense after DAMGO. Finally, NPFF(2) receptor activation was sufficient to recruit β-arrestin2 to the MOP receptor but not to induce its internalization. These data show that NPFF-induced heterologous desensitization of MOP receptor signaling is mediated by GRK2 and could involve transphosphorylation within the heteromeric receptor complex.

  4. Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons (United States)

    Scrogin, K. E.; Johnson, A. K.; Schmid, H. A.


    The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

  5. Adenosine A1 receptor-mediated transactivation of the EGF receptor produces a neuroprotective effect on cortical neurons in vitro

    Institute of Scientific and Technical Information of China (English)

    Ke-qiang XIE; Li-min ZHANG; Yan CAO; Jun ZHU; Lin-yin FENG


    Aim:To understand the mechanism of the transactivation of the epidermal growth factor receptor (EGFR) mediated by the adenosine A1 receptor (A1R).Methods:Primary cultured rat cortical neurons subjected to oxygen-glucose deprivation (OGD) and HEK293/A1R cells were treated with the A1R-specific agonist N6-cyclopentyladenosine (CPA).Phospho-EGFR,Akt,and ERK1/2 were observed by Western blot.An interaction between EGFR and AIR was detected using immunoprecipitation and immunocytochemistry.Results:The A1R agonist CPA causes protein kinase B (Akt) activation and protects primary cortical neurons from oxygen-glucose deprivation (OGD) insult.A1R and EGFR co-localize in the membranes of neurons and form an immunocomplex.A1R stimulation induces significant EGFR phosphorylation via a P13K and Src kinase signaling pathway;this stimulation provides a neuroprotective effect in cortical neurons.CPA leads to sustained phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) in cortical neurons,but only to transient phosphorylation in HEK 293/A1R cells.The response to the AtR agonist is mediated primarily through EGFR trans-activation that is dependent on pertussis toxin (PTX)-sensitive G1 protein and metalloproteases in HEK 293/A1R.Conclusion:A1R-mediated EGFR transactivation confers a neuroprotective effect in primary cortical neurons.P13 kinase and Src kinase play pivotal roles in this response.

  6. GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

    Institute of Scientific and Technical Information of China (English)

    Nariman A B Balenga; Maria Waldhoer; Elma Aflaki; Julia Kargl; Wolfgang Platzer; Ralf Schr(o)der; Stefanie Bl(a)ttermann; Evi Kostenis; Andrew J Brown; Akos Heinemann


    The directional migration of neutrophils towards inflammatory mediators,such as chemokines and cannabinoids,occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process.A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB2 receptor (CB2R),but additional modulatory sites distinct from CB2R have recently been suggested to impact CB2R-mediated effector functions in neutrophils.Here,we provide evidence that the recently de-orphanized 7TM/GPCR GPR55potently modulates CB2R-mediated responses.We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB2R agonist 2-arachidonoylglycerol (2-AG),while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production.Using HEK293 and HL60 cell lines,along with primary neutrophils,we show that GPR55 and CB2R interfere with each other's signaling pathways at the level of small GTPases,such as Rac2 and Cdc42.This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils.Therefore,GPR55 limits the tissueinjuring inflammatory responses mediated by CB2R,while it synergizes with CB2R in recruiting neutrophils to sites of inflammation.

  7. M1 muscarinic receptor activation mediates cell death in M1-HEK293 cells. (United States)

    Graham, E Scott; Woo, Kerhan K; Aalderink, Miranda; Fry, Sandie; Greenwood, Jeffrey M; Glass, Michelle; Dragunow, Mike


    HEK293 cells have been used extensively to generate stable cell lines to study G protein-coupled receptors, such as muscarinic acetylcholine receptors (mAChRs). The activation of M1 mAChRs in various cell types in vitro has been shown to be protective. To further investigate M1 mAChR-mediated cell survival, we generated stable HEK293 cell-lines expressing the human M1 mAChR. M1 mAChRs were efficiently expressed at the cell surface and efficiently internalised within 1 h by carbachol. Carbachol also induced early signalling cascades similar to previous reports. Thus, ectopically expressed M1 receptors behaved in a similar fashion to the native receptor over short time periods of analysis. However, substantial cell death was observed in HEK293-M1 cells within 24 h after carbachol application. Death was only observed in HEK cells expressing M1 receptors and fully blocked by M1 antagonists. M1 mAChR-stimulation mediated prolonged activation of the MEK-ERK pathway and resulted in prolonged induction of the transcription factor EGR-1 (>24 h). Blockade of ERK signalling with U0126 did not reduce M1 mAChR-mediated cell-death significantly but inhibited the acute induction of EGR-1. We investigated the time-course of cell death using time-lapse microscopy and xCELLigence technology. Both revealed the M1 mAChR cytotoxicity occurs within several hours of M1 activation. The xCELLigence assay also confirmed that the ERK pathway was not involved in cell-death. Interestingly, the MEK blocker did reduce carbachol-mediated cleaved caspase 3 expression in HEK293-M1 cells. The HEK293 cell line is a widely used pharmacological tool for studying G-protein coupled receptors, including mAChRs. Our results highlight the importance of investigating the longer term fate of these cells in short term signalling studies. Identifying how and why activation of the M1 mAChR signals apoptosis in these cells may lead to a better understanding of how mAChRs regulate cell-fate decisions.

  8. Amyloid β-protein differentially affects NMDA receptor- and GABAA receptor-mediated currents in rat hippocampal CA1 neurons

    Institute of Scientific and Technical Information of China (English)

    Junfang Zhang; Lei Hou; Xiuping Gao; Fen Guo; Wei Jing; Jinshun Qi; Jiantian Qiao


    Although the aggregated amyloid β-protein (Aβ) in senile plaques is one of the key neuropathological features of Alzheimer's disease (AD), soluble forms of Aβ also interfere with synaptic plasticity at the early stage of AD. The suppressive action of acute application of Aβ on hippocampal long-term potentiation (LTP) has been reported widely, whereas the mechanism underlying the effects of Aβ is still mostly unknown. The present study, using the whole-cell patch clamp technique, investigated the effects of Aβ fragments (Aβ25-35 and Aβ31-35) on the LTP induction-related postsynaptic ligand-gated channel currents in isolated hippocampal CA1 neurons. The results showed a rapid but opposite action of both peptides on excitatory and inhibitory receptor currents. Glutamate application-induced currents were suppressed by A β25-35 in a dose-dependent manner, and further N-methyl-I>aspartate (NMDA) receptor-mediated currents were selec-tively inhibited. In contrast, pretreatment with Aβ fragments potentiated γ-aminobutyric acid (GABA)-induced whole-cell currents. As a control, Aβ35-31 the reversed sequence of Aβ35-31 showed no effect on the currents induced by glutamate, NMDA or GABA. These results may partly explain the impaired effects of Aβ on hippocampal LTP, and suggest that the functional down-regulation of N M DA receptors and up-regulation of GABAA receptors may play an important role in remodeling the hippocampal synaptic plasticity in early AD.

  9. GR-127935-sensitive mechanism mediating hypotension in anesthetized rats: are 5-HT5B receptors involved? (United States)

    Sánchez-Maldonado, Carolina; López-Sánchez, Pedro; Anguiano-Robledo, Liliana; Leopoldo, Marcello; Lacivita, Enza; Terrón, José A


    The 5-HT1B/1D receptor antagonist, GR-127935, inhibits hypotensive responses produced by the 5-HT1A, 5-HT1B/1D and 5-HT7 receptor agonist, and 5-HT5A/5B receptor ligand, 5-carboxamidotryptamine (5-CT), in rats. This work further characterized the above mechanism using more selective 5-HT1B and 5-HT1D receptor antagonists. Also, expression of 5-HT5A and 5-HT5B receptor mRNAs in blood vessels was searched by reverse transcription polymerase chain reaction. Decreases in diastolic blood pressure induced by 5-CT (0.001-10 μg/kg, intravenously) were analyzed in anesthetized rats that had received intravenous vehicle (1 mL/kg), SB-224289 (5-HT1B antagonist; 0.3 and 1.0 mg/kg), BRL15572 (5-HT1D antagonist; 0.3 and 1.0 mg/kg), SB-224289 + BRL15572 (0.3 mg/kg, each), or SB-224289 + BRL15572 (0.3 mg/kg, each) + GR-127935 (1 mg/kg). Because only the latter treatment inhibited 5-CT-induced hypotension, suggestive of a mechanism unrelated to 5-HT1B/1D receptors, the effects of antagonists/ligands at 5-HT5A (SB-699551, 1 mg/kg), 5-HT6 (SB-399885, 1 mg/kg), and 5-HT1B/1D/5A/5B/7 receptors (ergotamine, 0.1 mg/kg) on 5-CT-induced hypotension were tested. Interestingly, only ergotamine blocked 5-CT-induced responses; this effect closely paralleled that of SB-224289 + BRL-15572 + GR-127935. Neither did ergotamine nor GR-127935 inhibit hypotensive responses induced by the 5-HT7 receptor agonist, LP-44. Faint but clear bands corresponding to 5-HT5A and 5-HT5B receptor mRNAs in aorta and mesenteric arteries were detected. Results suggest that the GR-127935-sensitive mechanism mediating hypotension in rats is unrelated to 5-HT1B, 5-HT1D, 5-HT5A, 5-HT6, and 5-HT7 receptors. This mechanism, however, resembles putative 5-HT5B receptors.

  10. Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells. (United States)

    Li, Qiang; Yan, Haidun; Wilson, Wilkie A; Swartzwelder, H Scott


    Although the endogenous cannabinoid system modulates a variety of physiological and pharmacological processes, the specific role of cannabinoid CB1 receptors in the modulation of glutamatergic neurotransmission and neural plasticity is not well understood. Using whole-cell patch clamp recording techniques, evoked or spontaneous excitatory postsynaptic currents (eEPSCs or sEPSCs) were recorded from visualized, layer II/III pyramidal cells in frontal cortical slices from rat brain. Bath application of the CB1 receptor agonist, WIN 55212-2 (WIN), reduced the amplitude of NMDA receptor-mediated EPSCs in a concentration-dependent manner. When co-applied with the specific CB1 antagonists, AM251 or AM281, WIN did not suppress NMDA receptor-mediated EPSCs. WIN also reduced the amplitude of evoked AMPA receptor-mediated EPSCs, an effect that was also reversed by AM251. Both the frequency and amplitude of spontaneous AMPA receptor-mediated EPSCs were significantly reduced by WIN. In contrast, WIN reduced the frequency, but not the amplitude of miniature EPSCs, suggesting that the suppression of glutamatergic activity by CB1 receptors in the frontal neocortex is mediated by a presynaptic mechanism. Taken together, these data indicate a critical role for endocannabinoid signaling in the regulation of excitatory synaptic transmission in frontal neocortex, and suggest a possible neuronal mechanism whereby THC regulates cortical function.

  11. Ganglioside mediate the interaction between Nogo receptor 1 and LINGO-1. (United States)

    Saha, Nayanendu; Kolev, Momchil V; Semavina, Mariya; Himanen, Juha; Nikolov, Dimitar B


    Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.

  12. Alpha adrenergic receptor mediation of cardiovascular and metabolic responses to alcohol

    Energy Technology Data Exchange (ETDEWEB)

    Brackett, D.J.; Gauvin, D.V.; Lerner, M.R.; Holloway, F.H.; Wilson, M.F. (Univ. of Oklahoma, Oklahoma City (United States) Veterans Affairs Medical Center, Oklahoma City, OK (United States))


    The role of alpha adrenergic receptors in acute cardiovascular and metabolic responses to alcohol (ETOH) have not been clearly defined. In this study two groups of male Sprague-Dawley rats were given intravenous phentolamine mesylate or saline prior to intragastric alcohol to blockade of alpha receptors during alcohol intoxication in conscious rats. ETOH alone evoked an increase in systemic vascular resistance (SVR), heart rate (HR), and blood glucose concentrations (G) and a decrease in mean arterial pressure (MAP), cardiac output (CO), central venous pressure (CVP), respiration rate (RR) and cardiac stroke volume (SV). Blood alcohol concentration (BAC) peaked at 30 min and remained elevated for the four hrs of monitoring. Phentolamine pretreatment produced a decrease in MAP and SV and an increase in HR. However, antagonism of the alpha receptor blocked the decrease in CO and the increase in SVR and G. The decrease in CVP was unaffected. Surprisingly, the early rise and peak in BAC in the phentolamine treated group was attenuated, but was the same as the untreated group during the final 3 hrs. These data suggest that alpha receptors are significant mediators of cardiovascular and glucoregulatory responses elicited by alcohol. Furthermore, alpha receptor blockade appears to effect the absorption and/or distribution of intragastrically administered alcohol.

  13. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition. (United States)

    Alari-Pahissa, Elisenda; Grandclément, Camille; Jeevan-Raj, Beena; Leclercq, Georges; Veillette, André; Held, Werner


    Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells' ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated "missing-self" reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.

  14. Serotonin(2) receptors mediate respiratory recovery after cervical spinal cord hemisection in adult rats. (United States)

    Zhou, S Y; Basura, G J; Goshgarian, H G


    The aim of the present study was to specifically investigate the involvement of serotonin [5-hydroxytryptamine (5-HT(2))] receptors in 5-HT-mediated respiratory recovery after cervical hemisection. Experiments were conducted on C(2) spinal cord-hemisected, anesthetized (chloral hydrate, 400 mg/kg ip), vagotomized, pancuronium- paralyzed, and artificially ventilated female Sprague-Dawley rats in which CO(2) levels were monitored and maintained. Twenty-four hours after spinal hemisection, the ipsilateral phrenic nerve displayed no respiratory-related activity indicative of a functionally complete hemisection. Intravenous administration of the 5-HT(2A/2C)-receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) induced respiratory-related activity in the phrenic nerve ipsilateral to hemisection under conditions in which CO(2) was maintained at constant levels and augmented the activity induced under conditions of hypercapnia. The effects of DOI were found to be dose dependent, and the recovery of activity could be maintained for up to 2 h after a single injection. DOI-induced recovery was attenuated by the 5-HT(2)-receptor antagonist ketanserin but not with the 5-HT(2C)-receptor antagonist RS-102221, suggesting that 5-HT(2A) and not necessarily 5-HT(2C) receptors may be involved in the induction of respiratory recovery after cervical spinal cord injury.

  15. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells. (United States)

    Olaso, E; Ikeda, K; Eng, F J; Xu, L; Wang, L H; Lin, H C; Friedman, S L


    Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

  16. Specific in vitro toxicity of crude and refined petroleum products. 1. Aryl hydrocarbon receptor-mediated responses

    NARCIS (Netherlands)

    Vrabie, C.M.; Jonker, M.T.O.; Murk, A.J.


    The present study is the first in a series reporting on in vitro toxic potencies of oils. The objective was to determine whether 11 crude oils and refined products activate the aryl hydrocarbon receptor (AhR) in a dioxin receptor¿mediated luciferase assay. Cells were exposed for 6 and 24 h to differ

  17. A novel gliotic P2 receptor mediating cyclooxygenase-2 induction in rat and human astrocytes. (United States)

    Brambilla, R; Ceruti, S; Malorni, W; Cattabeni, F; Abbracchio, M P


    In astrocytic cultures maintained in vitro, a brief challenge with the ATP analog alpha,beta methyleneATP (alpha,betameATP) results, 3 days later, in marked elongation of astrocytic processes, an event that resembles the astrocytic hypertrophy known to occur in vivo during reactive astrogliosis. alpha,beta meATP-induced effects were observed in primary astrocytes obtained from both rat striatum and cortex (a brain area highly involved in chronic neurodegenerative pathologies), as well as in human astrocytoma cells (ADF cells). Purine-induced gliosis could be reversed by the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS), but not by oxidized ATP (an antagonist of the P2X(7) receptor), in line with previous studies of our laboratory suggesting the involvement of a P2Y receptor subtype. Induction of reactive gliosis was preceded by increased expression of cyclooxygenase-2 (COX-2), an enzyme whose excessive activation has been implicated in both acute and chronic neurodegenerative diseases. The selective COX-2 inhibitor NS-398 prevented both purine-induced astrogliosis and the associated COX-2 induction, suggesting that inhibition of the transcription of the COX-2 gene may also contribute to the anti-inflammatory properties of this agent. Significant blockade of both alpha,beta meATP-mediated reactive gliosis and COX-2 induction was also observed with PPADS. These data suggest that COX-2 mediates P2Y receptor-induced reactive astrogliosis, and that antagonists selective for this receptor subtype may represent a novel class of anti-inflammatory agents of potential interest in acute and chronic neurological disorders characterized by an inflammatory component and reactive gliosis.

  18. Src, a molecular switch governing gain control of synaptic transmission mediated by N-methyl-d-aspartate receptors


    Yu, Xian-Min; Salter, Michael W


    The N-methyl-d-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activ...

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

    NARCIS (Netherlands)

    Gorvin, C.M.; Wilmer, M.J.G.; Piret, S.E.; Harding, B.; Heuvel, L.P.W.J. van den; Wrong, O.; Jat, P.S.; Lippiat, J.D.; Levtchenko, E.N.; Thakker, R.V.


    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, nephrolithias

  20. Delayed cardioprotection is mediated via a non-peptide delta opioid agonist, SNC-121, independent of opioid receptor stimulation. (United States)

    Patel, Hemal H; Hsu, Anna; Gross, Garrett J


    Acute cardioprotection is mediated primarily through delta opioid receptor stimulation independent of micro or kappa opioid receptor stimulation. Delayed cardioprotection is mediated by delta opioid receptor agonists but ambiguity remains about direct receptor involvement. Therefore, we investigated the potential of SNC-121, a non-peptide delta opioid agonist, to produce delayed cardioprotection and characterized the role of opioid receptors in this delayed response. All rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. SNC-121 induced a significant delayed cardioprotective effect. To determine the nature of this SNC-121-induced delayed cardioprotection, rats were treated with specific opioids receptor antagonists and underwent pertussis toxin (PT) treatment prior to opioid agonist stimulation. Control rats were injected with saline and allowed to recover for 24 hours. Pretreatment and early treatment with opioid receptor antagonists failed to inhibit the delayed protective effects of SNC-121, as did pretreatment with PT. Treatment with a free radical scavenger, 2-mercaptopropionyl glycine, at the time of opioid stimulation attenuated the delayed cardioprotective effects of SNC-121. These data suggest that delayed cardioprotection is stimulated via non-peptide delta opioid agonists by a mechanism unrelated to opioid receptor activation. The mechanism appears to be a non-opioid receptor mediated production of reactive oxygen species that triggers the signaling cascade leading to delayed cardioprotection.

  1. Fenugreek induced apoptosis in breast cancer MCF-7 cells mediated independently by fas receptor change. (United States)

    Alshatwi, Ali Abdullah; Shafi, Gowhar; Hasan, Tarique Noorul; Syed, Naveed Ahmed; Khoja, Kholoud Khalid


    Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently.

  2. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    Directory of Open Access Journals (Sweden)

    Samuel D Robinson


    Full Text Available NMDA receptors (NMDARs play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM but not high (50 μM concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-AP. Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and RAP, a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs.

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

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


    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.

  4. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

    LENUS (Irish Health Repository)

    Dooley, Ruth


    Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  5. Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

    LENUS (Irish Health Repository)

    Dooley, Ruth


    BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

  6. Neuropilin-1 mediates vascular permeability independently of vascular endothelial growth factor receptor-2 activation. (United States)

    Roth, Lise; Prahst, Claudia; Ruckdeschel, Tina; Savant, Soniya; Weström, Simone; Fantin, Alessandro; Riedel, Maria; Héroult, Mélanie; Ruhrberg, Christiana; Augustin, Hellmut G


    Neuropilin-1 (NRP1) regulates developmental and pathological angiogenesis, arteriogenesis, and vascular permeability, acting as a coreceptor for semaphorin 3A (Sema3A) and the 165-amino acid isoform of vascular endothelial growth factor A (VEGF-A165). NRP1 is also the receptor for the CendR peptides, a class of cell- and tissue-penetrating peptides with a specific R-x-x-R carboxyl-terminal motif. Because the cytoplasmic domain of NRP1 lacks catalytic activity, NRP1 is mainly thought to act through the recruitment and binding to other receptors. We report here that the NRP1 intracellular domain mediates vascular permeability. Stimulation with VEGF-A165, a ligand-blocking antibody, and a CendR peptide led to NRP1 accumulation at cell-cell contacts in endothelial cell monolayers, increased cellular permeability in vitro and vascular leakage in vivo. Biochemical analyses, VEGF receptor-2 (VEGFR-2) silencing, and the use of a specific VEGFR blocker established that the effects induced by the CendR peptide and the antibody were independent of VEGFR-2. Moreover, leakage assays in mice expressing a mutant NRP1 lacking the cytoplasmic domain revealed that this domain was required for NRP1-induced vascular permeability in vivo. Hence, these data define a vascular permeability pathway mediated by NRP1 but independent of VEGFR-2 activation.

  7. P2X7 Receptor Mediated Growth-Inhibitory Effect in KG1 a Cell Line

    Institute of Scientific and Technical Information of China (English)

    Xiujun Zhang; Lijun Meng


    This study was conducted to investigate ATP-induced growth inhibition in human leukemic cells KGla.METHoDS ATP inhibited cell growth was analyzed by MTSassay.Extemalization of phosphatidylserine could be detected byAnnexin-V-FITC apoptosis staining after activation of the P2X7 re-ceptor.P2X7 mediated pore formation was detected in KGla cellsby Yo-Pro-1 uptake assay.RESUlTS ATP inhibited cell growth in a dose-dependent man-ner.The cytotoxic effect could be blocked by P2X7 antagonists,oxidized ATP(OATP)and KN62.Externalization of phosphatidyl-serine could be detected in a time-dependent manner.P2X7 medi-ated pore forigation could be detected in KGla cells.These effectscould not be observed in P2X7 null Ramos cells.CONCLUSIoN The results and our previously reports thatmRNA,protein expression and calcium response of the P2X7receptor in KGla cells,suggested that extracellular ATP effectivelyinduces growth inhibition through apoptosis in KGla cells byactivation of P2X7 receptor,and that may be mediated by extracel-lular Ca2+ineux and pore formation.

  8. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor. (United States)

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki


    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

  9. Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons. (United States)

    Dumitriu, Bogdan; Cohen, Jonathan E; Wan, Qin; Negroiu, Andreea M; Abrams, Thomas W


    Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

  10. The anorectic effect of neurotensin is mediated via a histamine H1 receptor in mice. (United States)

    Ohinata, Kousaku; Shimano, Tomoko; Yamauchi, Rena; Sakurada, Shinobu; Yanai, Kazuhiko; Yoshikawa, Masaaki


    Neurotensin (NT), a tridecapeptide found in the mammalian brain and peripheral tissues, induces a decrease in food intake after central administration. In this investigation, we examine whether the histaminergic system is involved in NT-induced suppression of feeding. Intracerebroventricular injection of NT (0.1-1 nmol/mouse) led to dose-dependent inhibition of food intake in fasted ddY mice. The anorectic effect induced by NT (0.1 nmol/mouse) was ameliorated upon co-administration of pyrilamine (3 nmol/mouse), an antagonist for histomine H1 receptor. The NT-induced anorectic effect was partially ameliorated in H1 knockout mice. The findings suggest that the H1 receptor in part mediates the NT-induced suppression of food intake.

  11. Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

    Institute of Scientific and Technical Information of China (English)

    MingXU; Ying-huaGUAN; NingXU; Zhang-yiLIANG; Shu-yiWang; YaoSONG; Chi-deHAN; Xin-shengZHAO; You-yiZHANG


    AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell 3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.

  12. Subcellular localization of frizzled receptors, mediated by their cytoplasmic tails, regulates signaling pathway specificity.

    Directory of Open Access Journals (Sweden)

    Jun Wu


    Full Text Available The Frizzled (Fz; called here Fz1 and Fz2 receptors have distinct signaling specificities activating either the canonical Wnt/beta-catenin pathway or Fz/planar cell polarity (PCP signaling in Drosophila. The regulation of signaling specificity remains largely obscure. We show that Fz1 and Fz2 have different subcellular localizations in imaginal disc epithelia, with Fz1 localizing preferentially to apical junctional complexes, and Fz2 being evenly distributed basolaterally. The subcellular localization difference directly contributes to the signaling specificity outcome. Whereas apical localization favors Fz/PCP signaling, it interferes with canonical Wnt/beta-catenin signaling. Receptor localization is mediated by sequences in the cytoplasmic tail of Fz2 that appear to block apical accumulation. Based on these data, we propose that subcellular Fz localization, through the association with other membrane proteins, is a critical aspect in regulating the signaling specificity within the Wnt/Fz signaling pathways.

  13. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat. (United States)

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


    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.

  14. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells (United States)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.


    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  15. β-arrestins: regulatory role and therapeutic potential in opioid and cannabinoid receptor-mediated analgesia. (United States)

    Raehal, Kirsten M; Bohn, Laura M


    Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of "pharmacological" interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects.

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

    Directory of Open Access Journals (Sweden)

    Luigi Bozzo

    Full Text Available 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.

  17. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway. (United States)

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


    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.

  18. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression (United States)

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas


    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  19. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor. (United States)

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen


    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

  20. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

    Directory of Open Access Journals (Sweden)

    Ana Talamillo


    Full Text Available SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor, the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

  1. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor. (United States)

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M


    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  2. Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice. (United States)

    McVicker, Benita L; Thiele, Geoffrey M; Casey, Carol A; Osna, Natalia A; Tuma, Dean J


    T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

  3. P2X7 receptors mediate innate phagocytosis by human neural precursor cells and neuroblasts. (United States)

    Lovelace, Michael D; Gu, Ben J; Eamegdool, Steven S; Weible, Michael W; Wiley, James S; Allen, David G; Chan-Ling, Tailoi


    During early human neurogenesis there is overproduction of neuroblasts and neurons accompanied by widespread programmed cell death (PCD). While it is understood that CD68(+) microglia and astrocytes mediate phagocytosis during target-dependent PCD, little is known of the cell identity or the scavenger molecules used to remove apoptotic corpses during the earliest stages of human neurogenesis. Using a combination of multiple-marker immunohistochemical staining, functional blocking antibodies and antagonists, we showed that human neural precursor cells (hNPCs) and neuroblasts express functional P2X7 receptors. Furthermore, using live-cell imaging, flow cytometry, phagocytic assays, and siRNA knockdown, we showed that in a serum-free environment, doublecortin(+) (DCX) neuroblasts and hNPCs can clear apoptotic cells by innate phagocytosis mediated via P2X7. We found that both P2X7(high) DCX(low) hNPCs and P2X7(high) DCX(high) neuroblasts, derived from primary cultures of human fetal telencephalon, phagocytosed targets including latex beads, apoptotic ReNcells, and apoptotic hNPC/neuroblasts. Pretreatment of neuroblasts and hNPCs with 1 mM adenosine triphosphate (ATP), 100 µM OxATP (P2X7 antagonist), or siRNA knockdown of P2X7 inhibited phagocytosis of these targets. Our results show that P2X7 functions as a scavenger receptor under serum-free conditions resembling those in early neurogenesis. This is the first demonstration that hNPCs and neuroblasts may participate in clearance of apoptotic corpses during pre target-dependent neurogenesis and mediate phagocytosis using P2X7 as a scavenger receptor.

  4. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J


    , and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism......The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR...... phosphorylation could inhibit PKC-catalyzed heterologous DOR phosphorylation and subsequent internalization. These data demonstrate that the responsiveness of opioid receptor is regulated by both PKC and GRK through agonist-dependent and agonist-independent mechanisms and PKC-mediated receptor phosphorylation...

  5. α1A-adrenergic receptor mediated pressor response to phenylephrine in anesthetized rat

    Institute of Scientific and Technical Information of China (English)

    XU Qi; ZHU Weizhong; L(U) Zhizhen; ZHANG Youyi; HAN Qide


    To determine which subtype of α1A-adrenergic receptors plays a role in the regulation of blood pressure, with α1A-adrenergic receptor-mediated vasoconstriction in perfused hindlimb as a control, we compared the inhibitory effects of various α1A-adrenergic receptor selective antagonists on the vasopressure responses to phenylephrine between the mean arterial pressure and hindlimb perfusion pressure in anesthetized rats. In Normotensive Wistar rats, the results showed that the inhibitory effects (dose ratios of ED50, Dr) of α1A-adrenoceptor selective antagonist (prazosin, Dr 13.5 ± 3.6 vs.15.1 ± 4.3, n = 11), α1A-adrenoceptor selective antagonist (5- methyl-urapidil, Dr 2.4 ± 0.9 vs. 3.7 ± 2.3, n = 12; RS-17053, Dr 3.2 ± 1.6 vs. 4.4 ± 3.3, n =12) and α1D- adrenoceptor selective antagonist (BMY7378, Dr 1.9 ± 0.9 vs. 2.2 ± 0.8, n = 8) on phenylephrine- induced increases of perfusion pressure in the autoperfused femoral beds were the same as that in the mean arterial blood pressure in normotensive Wistar rats. The inhibitory effects of antagonists (RS-17053, Dr 3.4 ± 0.6 vs. 4.3 ± 0.9, n = 5; BMY7378, Dr 1.7±0.5 vs. 1.7 ± 0.5, n = 8) in spontaneous hypertensive rats were similar with the Wistar rats. These results suggest that the mean arterial pressure induced by phenylephrine was mainly mediated by α1A-adrenergic receptor in both the anesthetized Wistar rats and spontaneous hypertensive rats.

  6. The CB1 receptor as an important mediator of hedonic reward processing. (United States)

    Friemel, Chris M; Zimmer, Andreas; Schneider, Miriam


    The endocannabinoid (ECB) system has emerged recently as a key mediator for reward processing. It is well known that cannabinoids affect appetitive learning processes and can induce reinforcing and rewarding effects. However, the involvement of the ECB system in hedonic aspects of reward-related behavior is not completely understood. With the present study, we investigated the modulatory role of the ECB system on hedonic perception, measured by the pleasure attenuated startle (PAS) paradigm for a palatable food reward. Here, a conditioned odor is thought to induce a pleasant affective state that attenuates an aversive reflex-the acoustic startle response. Modulatory effects of the CB1 receptor antagonist/inverse agonist SR1411716 and the cannabinoid agonist WIN 55 212-2 on PAS were examined in rats. PAS was also measured in CB1 receptor knockout (KO) and wild-type (WT) mice. Pharmacological inhibition as well as the absence of CB1 receptors was found to reduce PAS, whereas WIN 55 212-2 administration increased PAS. Finally, presentation of a conditioned reward cue was found to induce striatal FosB/ΔFosB expression in WT mice, but not in KO mice, indicating a reduced stimulation of reward-related brain regions in conditioned KO mice by odor presentation. We here show that in addition to our previous studies in rats, PAS may also serve as a valuable and suitable measure to assess hedonic processing in mice. Our data further indicate that the ECB system, and in particular CB1 receptor signaling, appears to be highly important for the mediation of hedonic aspects of reward processing.

  7. Neuronal nicotinic acetylcholine receptors serve as sensitive targets that mediate β-amyloid neurotoxicity

    Institute of Scientific and Technical Information of China (English)

    Qiang LIU; Jie WU


    Alzheimer's disease (AD) is the most common form of brain dementia characterized by the accumulation of β-amyloid peptides (Aβ) and loss of forebrain cholinergic neurons. Aβ accumulation and aggregation are thought to contribute to cholinergic neuronal degeneration, in turn causing learning and memory deficits, but the specific targets that mediate Aβ neurotoxicity remain elusive. Recently, accumlating lines of evidence have demonstrated that Aβ directly modulates the function of neuronal nicotinic acetylcholine receptors (nAChRs), which leads to the new hypothesis that neuronal nAChRs may serve as important targets that mediate Aβ neurotoxicity. In this review, we summarize current studies performed in our laboratory and in others to address the question of how Aβ modulates neuronal nAChRs, especially nAChR subunit function.

  8. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse. (United States)

    Gocel, James; Larson, John


    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  9. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis. (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K


    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  10. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation. (United States)

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane


    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved.

  11. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity. (United States)

    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D


    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  12. Vitamin D Receptor-Mediated Upregulation of CYP3A4 and MDR1 by Quercetin in Caco-2 cells. (United States)

    Chae, Yoon-Jee; Cho, Kwan Hyung; Yoon, In-Soo; Noh, Chi-Kyoung; Lee, Hyo-Jong; Park, Yohan; Ji, Eunhee; Seo, Min-Duk; Maeng, Han-Joo


    To examine whether quercetin interacts with vitamin D receptor, we investigated the effects of quercetin on vitamin D receptor activity in human intestinal Caco-2 cells. The effects of quercetin on the expression of the vitamin D receptor target genes, vitamin D3 24-hydroxylase, cytochrome P450 3A4, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were measured using quantitative polymerase chain reaction. The vitamin D receptor siRNA was used to assess the involvement of the vitamin D receptor. Vitamin D receptor activation using a vitamin D responsive element-mediated cytochrome P450 3A4 reporter gene assay was investigated in Caco-2 cells transfected with human vitamin D receptor. We also studied the magnitude of the vitamin D receptor activation and/or synergism between 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] and quercetin-like flavonoids. Slight but significant increases in the mRNA expression of cytochrome P450 3A4, vitamin D3 24-hydroxylase, multidrug resistance protein 1, and transient receptor potential vanilloid type 6 were observed after 3 days of continual quercetin treatment. The silencing effect of vitamin D receptor by vitamin D receptor siRNA in Caco-2 cells significantly attenuated the induction of the vitamin D receptor target genes. Moreover, quercetin significantly enhanced cytochrome P450 3A4 reporter activity in Caco-2 cells in a dose-dependent manner, and the expression of exogenous vitamin D receptor further stimulated the vitamin D receptor activity. Quercetin-like flavonoids such as kaempferol stimulated the vitamin D receptor activity in a manner similar to that seen with quercetin. Taken together, the data indicates that quercetin upregulates cytochrome P450 3A4 and multidrug resistance protein 1 expression in Caco-2 cells likely via a vitamin D receptor-dependent pathway.

  13. Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays. (United States)

    Ghosh, Madhubrata; Wang, Loo Chien; Ramesh, Ranita; Morgan, Leslie K; Kenney, Linda J; Anand, Ganesh S


    Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

  14. Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin. (United States)

    Gburek, Jakub; Birn, Henrik; Verroust, Pierre J; Goj, Bogusława; Jacobsen, Christian; Moestrup, Søren K; Willnow, Thomas E; Christensen, Erik I


    Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kd values of 2.0 and 3 microM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced (Kd = 5 microM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes (Ki approximately 2 microM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.

  15. Cardio-respiratory effects of systemic neurotensin injection are mediated through activation of neurotensin NTS₁ receptors. (United States)

    Kaczyńska, Katarzyna; Szereda-Przestaszewska, Małgorzata


    The purpose of our study was to determine the cardio-respiratory pattern exerted by the systemic injection of neurotensin, contribution of neurotensin NTS(1) receptors and the neural pathways mediating the responses. The effects of an intravenous injection (i.v.) of neurotensin were investigated in anaesthetized, spontaneously breathing rats in following experimental schemes: (i) control animals before and after midcervical vagotomy; (ii) in three separate subgroups of rats: neurally intact, vagotomized at supranodosal level and initially midcervically vagotomized exposed to section of the carotid sinus nerves (CSNs); (iii) in the intact rats 2 minutes after blockade of neurotensin NTS(1) receptors with SR 142948. Intravenous injection of 10 μg/kg of neurotensin in the intact rats evoked prompt increase in the respiratory rate followed by a prolonged slowing down coupled with augmented tidal volume. Midcervical vagotomy precluded the effects of neurotensin on the frequency of breathing, while CSNs section reduced the increase in tidal volume. In all the neural states neurotensin caused significant fall in mean arterial blood pressure preceded by prompt hypertensive response. The cardio-respiratory effects of neurotensin were blocked by pre-treatment with NTS(1) receptor antagonist. The results of this study showed that neurotensin acting through NTS(1) receptors augments the tidal component of the breathing pattern in a large portion via carotid body afferentation whereas the respiratory timing response to neurotensin depends entirely on the intact midcervical vagi. Blood pressure effects evoked by an intravenous neurotensin occur outside vagal and CSNs pathways and might result from activation of the peripheral vascular NTS(1) receptors.

  16. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

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    Jennes, L.; Stumpf, W.E.; Conn, P.M.


    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.

  17. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis. (United States)

    Richards, David M; Endres, Robert G


    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.

  18. Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor. (United States)

    Chen, Keyang; Williams, Kevin Jon


    Endocytosis via rafts has attracted considerable recent interest, but the molecular mediators remain incompletely characterized. Here, we focused on the syndecan-1 heparan sulfate proteoglycan, a highly conserved, multifunctional receptor that we previously showed to undergo raft-dependent endocytosis upon clustering. Alanine scanning mutagenesis of three to five consecutive cytoplasmic residues at a time revealed that a conserved juxtamembrane motif, MKKK, was the only region required for efficient endocytosis after clustering. Endocytosis of clustered syndecan-1 occurs in two phases, each requiring a kinase and a corresponding cytoskeletal partner. In the initial phase, ligands trigger rapid MKKK-dependent activation of ERK and the localization of syndecan-1 into rafts. Activation of ERK drives the dissociation of syndecan-1 from α-tubulin, a molecule that may act as an anchor for syndecan-1 at the plasma membrane in the basal state. In the second phase, Src family kinases phosphorylate tyrosyl residues within the transmembrane and cytoplasmic regions of syndecan-1, a process that also requires MKKK. Tyrosine phosphorylation of syndecan-1 triggers the robust recruitment of cortactin, which we found to be an essential mediator of efficient actin-dependent endocytosis. These findings represent the first detailed characterization of the molecular events that drive endocytosis of a raft-dependent receptor and identify a novel endocytic motif, MKKK. Moreover, the results provide new tools to study syndecan function and regulation during uptake of its biologically and medically important ligands, such as HIV-1, atherogenic postprandial remnant lipoproteins, and molecules implicated in Alzheimer disease.

  19. Erk1/2 mediates leptin receptor signaling in the ventral tegmental area.

    Directory of Open Access Journals (Sweden)

    Richard Trinko

    Full Text Available Leptin acts on the ventral tegmental area (VTA to modulate neuronal function and feeding behavior in rats and mice. To identify the intracellular effectors of the leptin receptor (Lepr, downstream signal transduction events were assessed for regulation by direct leptin infusion. Phosphorylated signal transducer and activator of transcription 3 (pSTAT3 and phosphorylated extracellular signal-regulated kinase-1 and -2 (pERK1/2 were increased in the VTA while phospho-AKT (pAKT was unaffected. Pretreatment of brain slices with the mitogen-activated protein kinase kinase -1 and -2 (MEK1/2 inhibitor U0126 blocked the leptin-mediated decrease in firing frequency of VTA dopamine neurons. The anorexigenic effects of VTA-administered leptin were also blocked by pretreatment with U0126, which effectively blocked phosphorylation of ERK1/2 but not STAT3. These data demonstrate that pERK1/2 may have a critical role in mediating both the electrophysiogical and behavioral effects of leptin receptor signaling in the VTA.

  20. Benzodiazepine receptor-mediated behavioral effects of nitrous oxide in the rat social interaction test. (United States)

    Quock, R M; Wetzel, P J; Maillefer, R H; Hodges, B L; Curtis, B A; Czech, D A


    The present study was conducted to ascertain whether an anxiolytic effect of nitrous oxide was demonstrable in rats using the social interaction test and whether this drug effect might be mediated by benzodiazepine receptors. Compared to behavior of vehicle-pretreated, room air-exposed rats, rat pairs exposed to nitrous oxide showed a generally inverted U-shaped dose-response curve with the maximum increase in social interaction encounters occurring at 25% and significant increase in time of active social interaction at 15-35%; higher concentrations produced a sedative effect that reduced social interaction. Treatment with 5.0 mg/kg of the anxiolytic benzodiazepine chlordiazepoxide also increased social interaction. Pretreatment with 10 mg/kg of the benzodiazepine receptor blocker flumazenil, which alone had no effect, significantly antagonized the social interaction-increasing effects of both nitrous oxide and chlordiazepoxide. In summary, these findings suggest that nitrous oxide produces a flumazenil-sensitive effect comparable to that of chlordiazepoxide and implicate central benzodiazepine mechanisms in mediation of the anxiolytic effect of nitrous oxide.

  1. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria

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    E.V. Seliverstova


    Full Text Available The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes, and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals.

  2. Receptor-Mediated and Fluid-Phase Transcytosis of Horseradish Peroxidase across Rat Hepatocytes

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    Isabella Ellinger


    Full Text Available Horseradish peroxidase (HRP is often used as a fluid-phase marker to characterize endocytic and transcytotic processes. Likewise, it has been applied to investigate the mechanisms of biliary secretion of fluid in rat liver hepatocytes. However, HRP contains mannose residues and thus binds to mannose receptors (MRs on liver cells, including hepatocytes. To study the role of MR-mediated endocytosis of HRP transport in hepatocytes, we determined the influence of the oligosaccharid mannan on HRP biliary secretion in the isolated perfused rat liver. A 1-minute pulse of HRP was applied followed by marker-free perfusion. HRP appeared in bile with biphasic kinetics: a first peak at 7 minutes and a second peak at 15 minutes after labeling. Perfusion with 0.8 mg/mL HRP in the presence of a twofold excess of mannan reduced the first peak by 41% without effect on the second one. Together with recently published data on MR expression in rat hepatocytes this demonstrates two different mechanisms for HRP transcytosis: a rapid, receptor-mediated transport and a slower fluid-phase transport.

  3. Estrogen receptor-mediated transcription involves the activation of multiple kinase pathways in neuroblastoma cells. (United States)

    Clark, Sara; Rainville, Jennifer; Zhao, Xing; Katzenellenbogen, Benita S; Pfaff, Donald; Vasudevan, Nandini


    While many physiological effects of estrogens (E) are due to regulation of gene transcription by liganded estrogen receptors (ERs), several effects are also mediated, at least in part, by rapid non-genomic actions of E. Though the relative importance of rapid versus genomic effects in the central nervous system is controversial, we showed previously that membrane-limited effects of E, initiated by an estradiol bovine serum albumin conjugate (E2-BSA), could potentiate transcriptional effects of 17β-estradiol from an estrogen response element (ERE)-reporter in neuroblastoma cells. Here, using specific inhibitors and activators in a pharmacological approach, we show that activation of phosphatidylinositol-3-phosphate kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways, dependent on a Gαq coupled receptor signaling are important in this transcriptional potentiation. We further demonstrate, using ERα phospho-deficient mutants, that E2-BSA mediated phosphorylation of ERα is one mechanism to potentiate transcription from an ERE reporter construct. This study provides a possible mechanism by which signaling from the membrane is coupled to transcription in the nucleus, providing an integrated view of hormone signaling in the brain.

  4. Nicotine alpha 4 beta 2 receptor-mediated free calcium in an animal model of facial nucleus injury

    Institute of Scientific and Technical Information of China (English)

    Dawei Sun; Wenhai Sun; Yanqing Wang; Fugao Zhu; Rui Zhou; Yanjun Wang; Banghua Liu; Xiuming Wan; Huamin Liu


    Previous studies have demonstrated that the cholinergic system,via nicotinic receptors,regulates intracellular free calcium levels in the facial nucleus under normal physiological conditions.However,the regulation of nicotinic receptors on free calcium levels following facial nerve injury remains unclear.In the present study,an animal model of facial nerve injury was established,and changes in nicotinic receptor expression following facial nerve injury in rats were detected using reverse transcription polymerase chain reaction.Nicotinic receptor-mediated changes of free calcium levels following facial nucleus injury were determined by laser confocal microscopy.Results showed no significant difference in nicotinic receptor expression between the normal group and the affected facial nerve nucleus.The nicotinic receptor α4β2 subtype increased free calcium levels following facial nerve injury by promoting calcium transmembrane influx,and L-type voltage-gated calcium channel-mediated influx of calcium ions played an important role in promoting calcium transmembrane influx.The nicotinic receptor-mediated increase of free calcium levels following facial nerve injury provides an important mechanism for the repair of facial nerve injury.

  5. Carrier-mediated release of monoamines induced by the nicotinic acetylcholine receptor agonist DMPP. (United States)

    Szász, Bernadett K; Mayer, Aliz; Zsilla, Gabriella; Lendvai, Balázs; Vizi, E Sylvester; Kiss, János P


    We have previously shown that dimethylphenylpiperazinium (DMPP) increases the release of noradrenaline (NA) from rat hippocampal slices via two distinct mechanisms: a nicotinic acetylcholine receptor (nAChR)-mediated exocytosis and a carrier-mediated release induced by the reversal of NA transporters. Our aim was to investigate whether other monoaminergic systems are also affected by the multiple actions of DMPP. In our experiments DMPP dose-dependently increased the release of dopamine (DA) and serotonin (5-HT) from rat striatal and hippocampal slices, respectively. The dual effect was observed, however, only in case of DA at a lower DMPP concentration (30 microM), where the response was partly inhibited by mecamylamine, TTX and Ca2+-free medium (nAChR-mediated exocytosis) while the other part of the response was blocked only by the DA uptake inhibitor nomifensine (carrier-mediated release). In contrast, the DMPP-evoked 5-HT release and the DA release induced by high concentration DMPP was not inhibited by nicotinic antagonists, TTX and Ca2+-free medium but only by selective uptake inhibitors. In addition, DMPP dose-dependently inhibited the [3H]DA and [3H]5-HT uptake in striatal and hippocampal synaptosome preparation with an IC50 of 3.18 and 0.49 microM, respectively. Our data show that DMPP interacts with monoamine transporters and induces a substantial carrier-mediated release of DA and 5-HT, therefore caution is needed for the interpretation of data, when this drug is used as a nAChR agonist.

  6. Receptor subtype involved in α1-adrenergic receptor-mediated Ca2+ sig-naling in cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Da-li LUO; Jian GAO; Lin-lin FAN; Yu TANG; You-yi ZHANG; Qi-de HAN


    Aim: The enhancement of intracellular Ca2+ signaling in response to α1-adrener-gic receptor (α1-AR) stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role(s) of the α1-AR subtype(s) in mediating this response. Methods: We evaluated the effects of subtype-specific agonists and antagonists of the α1- AR on the intracellular Ca2+ signaling of neonatal rat ventricular myocytes using a confocal microscope. Results: After being cultured for 48 h, the myocytes exhibited spontaneous local Ca2+ release, sparks, and global Ca2+ transients. The activation of the α1-AR with phenylephrine, a selective agonist of the α1-AR, dose-dependently increased the frequency of Ca2+ transients with an EC50 value of 2.3 μmol/L. Blocking the α1A-AR subtype with 5-methyhirapidil (5-Mu) inhi-bited the stimulatory effect of phenylephrine with an IC50 value of 6.7 nmol/L. In contrast, blockade of the α1B-AR and α1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca2+ spark numbers were also increased by the activation of theα1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective α1A-AR agonist, mimicked the effects of phenylephrine, but with more potency (EC50 value =6.9 nmol/L) in the potentiation of Ca2+ transients, and blockade of the α1A-AR by 5-Mu caused abolishment of its effects. Conclusion: These results indicate that α1-adrenergic stimulation of intracellular Ca2+ activity is mediated selectively by the α1A-AR.

  7. Probing multivalency in ligand–receptor-mediated adhesion of soft, biomimetic interfaces

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    Stephan Schmidt


    Full Text Available Many biological functions at cell level are mediated by the glycocalyx, a dense carbohydrate-presenting layer. In this layer specific interactions between carbohydrate ligands and protein receptors are formed to control cell–cell recognition, cell adhesion and related processes. The aim of this work is to shed light on the principles of complex formation between surface anchored carbohydrates and receptor surfaces by measuring the specific adhesion between surface bound mannose on a concanavalin A (ConA layer via poly(ethylene glycol-(PEG-based soft colloidal probes (SCPs. Special emphasis is on the dependence of multivalent presentation and density of carbohydrate units on specific adhesion. Consequently, we first present a synthetic strategy that allows for controlled density variation of functional groups on the PEG scaffold using unsaturated carboxylic acids (crotonic acid, acrylic acid, methacrylic acid as grafting units for mannose conjugation. We showed by a range of analytic techniques (ATR–FTIR, Raman microscopy, zeta potential and titration that this synthetic strategy allows for straightforward variation in grafting density and grafting length enabling the controlled presentation of mannose units on the PEG network. Finally we determined the specific adhesion of PEG-network-conjugated mannose units on ConA surfaces as a function of density and grafting type. Remarkably, the results indicated the absence of a molecular-level enhancement of mannose/ConA interaction due to chelate- or subsite-binding. The results seem to support the fact that weak carbohydrate interactions at mechanically flexible interfaces hardly undergo multivalent binding but are simply mediated by the high number of ligand–receptor interactions.

  8. Probing multivalency in ligand–receptor-mediated adhesion of soft, biomimetic interfaces (United States)

    Schmidt, Stephan; Wang, Hanqing; Pussak, Daniel; Mosca, Simone


    Summary Many biological functions at cell level are mediated by the glycocalyx, a dense carbohydrate-presenting layer. In this layer specific interactions between carbohydrate ligands and protein receptors are formed to control cell–cell recognition, cell adhesion and related processes. The aim of this work is to shed light on the principles of complex formation between surface anchored carbohydrates and receptor surfaces by measuring the specific adhesion between surface bound mannose on a concanavalin A (ConA) layer via poly(ethylene glycol)-(PEG)-based soft colloidal probes (SCPs). Special emphasis is on the dependence of multivalent presentation and density of carbohydrate units on specific adhesion. Consequently, we first present a synthetic strategy that allows for controlled density variation of functional groups on the PEG scaffold using unsaturated carboxylic acids (crotonic acid, acrylic acid, methacrylic acid) as grafting units for mannose conjugation. We showed by a range of analytic techniques (ATR–FTIR, Raman microscopy, zeta potential and titration) that this synthetic strategy allows for straightforward variation in grafting density and grafting length enabling the controlled presentation of mannose units on the PEG network. Finally we determined the specific adhesion of PEG-network-conjugated mannose units on ConA surfaces as a function of density and grafting type. Remarkably, the results indicated the absence of a molecular-level enhancement of mannose/ConA interaction due to chelate- or subsite-binding. The results seem to support the fact that weak carbohydrate interactions at mechanically flexible interfaces hardly undergo multivalent binding but are simply mediated by the high number of ligand–receptor interactions. PMID:26124875

  9. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells. (United States)

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


    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  10. Nicotinic receptor mediates nitric oxide synthase expression in the rat gastric myenteric plexus. (United States)

    Nakamura, K; Takahashi, T; Taniuchi, M; Hsu, C X; Owyang, C


    The mechanism that regulates the synthesis of nitric oxide synthase (NOS), a key enzyme responsible for NO production in the myenteric plexus, remains unknown. We investigated the roles of the vagal nerve and nicotinic synapses in the mediation of NOS synthesis in the gastric myenteric plexus in rats. Truncal vagotomy and administration of hexamethonium significantly reduced nonadrenergic, noncholinergic relaxation, the catalytic activity of NOS, the number of NOS-immunoreactive cells, and the density of NOS-immunoreactive bands and NOS mRNA bands obtained from gastric tissue. These results suggest that NOS expression in the gastric myenteric plexus is controlled by the vagal nerve and nicotinic synapses. We also investigated if stimulation of the nicotinic receptor increases neuronal NOS (nNOS) expression in cultured gastric myenteric ganglia. Incubation of cultured gastric myenteric ganglia with the nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperizinium (DMPP, 10(-10)-10(-7) M), for 24 h significantly increased the number of nNOS-immunoreactive cells and the density of immunoreactive nNOS bands and nNOS mRNA bands. nNOS mRNA expression stimulated by DMPP was antagonized by a protein kinase C antagonist, a phospholipase C inhibitor, and an intracellular Ca2+ chelator. We concluded that activation of the nicotinic receptor stimulates a Ca2+-dependent protein kinase C pathway, which in turn, upregulates nNOS mRNA expression and nNOS synthesis in the gastric myenteric plexus.

  11. The Melanocortin 3 Receptor: A Novel Mediator of Exercise-Induced Inflammation Reduction in Postmenopausal Women?

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    Tara M. Henagan


    Full Text Available The purpose of this study was to determine whether resistance exercise training-induced reductions in inflammation are mediated via melanocortin 3 receptor expression in obese (BMI 32.7±3.7 women (65.6±2.8 yrs randomized to either a control (=11 or resistance training group (=12. The resistance trained group performed resistance training 3 days/week for 12 weeks. Resting blood samples were collected before and after the training intervention in both resistance trained and control groups. Resistance training upregulated melanocortin 3 receptor mRNA by 16-fold (=.035 and decreased monocyte count, without changing leukocyte number, body composition, or body weight. Resistance trained individuals exhibited increased sensitivity to inflammatory stimuli, whereas control individuals exhibited no change. While there was no change in whole blood tumor necrosis factor alpha mRNA between the groups, whole blood interleukin 10 mRNA was higher in the resistance trained group following the intervention period. In summary, it appears that resistance training may modulate melanocortin 3 receptor expression, providing a possible mechanism for the anti-inflammatory effects of exercise training.

  12. Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1. (United States)

    Robinson, Jessica L L; Macarthur, Stewart; Ross-Innes, Caryn S; Tilley, Wayne D; Neal, David E; Mills, Ian G; Carroll, Jason S


    Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

  13. P2X7 receptors and Fyn kinase mediate ATP-induced oligodendrocyte progenitor cell migration. (United States)

    Feng, Ji-Feng; Gao, Xiao-Fei; Pu, Ying-Yan; Burnstock, Geoffrey; Xiang, Zhenghua; He, Cheng


    Recruitment of oligodendrocyte precursor cells (OPCs) to the lesions is the most important event for remyelination after central nervous system (CNS) injury or in demyelinating diseases. However, the underlying molecular mechanism is not fully understood. In the present study, we found high concentrations of ATP could increase the number of migrating OPCs in vitro, while after pretreatment with oxidized ATP (a P2X7 receptor antagonist), the promotive effect was attenuated. The promotive effect of 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) (a P2X7 receptor agonist) was more potent than ATP. After incubation with BzATP, the activity of Fyn, one member of the Src family of kinases, was enhanced. Moreover, the interaction between P2X7 and Fyn was identified by co-immunoprecipitation. After blocking the activity of Fyn or down-regulating the expression of Fyn, the migration of OPCs induced by BzATP was inhibited. These data indicate that P2X7 receptors/Fyn may mediate ATP-induced OPC migration under pathological conditions.

  14. Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons. (United States)

    Booker, Sam A; Gross, Anna; Althof, Daniel; Shigemoto, Ryuichi; Bettler, Bernhard; Frotscher, Michael; Hearing, Matthew; Wickman, Kevin; Watanabe, Masahiko; Kulik, Ákos; Vida, Imre


    Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.

  15. Regulative effect of anandamide-mediated cannabinoid receptor in rats with visceral hypersensitivity

    Directory of Open Access Journals (Sweden)

    Yu-qin HE


    Full Text Available Objective  To investigate the role of anandamide(ANA-mediated cannabinoid receptor 1(CB1 on the acquisition of visceral hypersensitivity in rats, and explore its underlying mechanism. Methods  The visceral hypersensitivity non-noxious/noxious colorectal distension (NNCRD/NCRD model of rat was reproduced by ovalbumin (OVA sensitization combined with NNCRD/NCRD. Fifty-four rats were randomly divided into control group (n=7, saline+CRD group (n=7, OVA+CRD+dimethyl sulfoxide (DMSO group (n=8, OVA+CRD+different concentrations of ANA (0.5, 5.0, 10.0mg/kg groups (8 each, and OVA+CRD+ANA+AM251 group (n=8. The expression and quantitative assessment of CB1 were monitored by immunoflurorescence and laser scanning confocal analysis. The visceral sensitivity was evaluated by the area under curve (AUC of myoelectrical activity of abdominal wall muscle. Results  By NCRD at 80mmHg, the density of CB1 immunofluorescence intensity was significantly higher in L4–L6 of the spinal cord of the rats in saline+CRD group compared with that in control group (P 0.05. By NCRD at 80mmHg, the VMR-AUC increased obviously in OVA+CRD+DMSO group as compared with that of saline+CRD group, but it decreased significantly in OVA+CRD+high concentration ANA group (P < 0.05. When AM251 was intravenously given, VMR-AUC increased significantly in OVA+CRD+ANA+AM251 group compared with that in OVA+CRD+different concentrations of ANA groups (P < 0.05. Conclusions Intravenous administration of ANA may mitigate the visceral nociception induced by basic OVAsensitization combined with NCRD stimulation in CB1-mediated manner. It indicated that anandamide-mediated CB1 cannabinoid receptor may regulate the development and maintenance of visceral hypersensitivity.

  16. Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells. (United States)

    Lajeunesse, Francis; Kröger, Helmut; Timofeev, Igor


    Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-D-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size.

  17. P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice. (United States)

    Chatterjee, Saurabh; Rana, Ritu; Corbett, Jean; Kadiiska, Maria B; Goldstein, Joyce; Mason, Ronald P


    While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

  18. Receptor-mediated activation of gastric vagal afferents by glucagon-like peptide-1 in the rat

    DEFF Research Database (Denmark)

    Bucinskaite, V; Tolessa, T; Pedersen, J


    The vagus nerve plays a role in mediating effects of the two glucagon-like peptides GLP-1 and GLP-2 on gastrointestinal growth, functions and eating behaviour. To obtain electrophysiological and molecular evidence for the contribution of afferent pathways in chemoreception from the gastrointestinal...... tract, afferent mass activity in the ventral gastric branch of the vagus nerve and gene expression of GLP-1 receptors and GLP-2 receptors in the nodose ganglion were examined in Sprague-Dawley rats. Intravenous administration of GLP-1 (30-1000 pmol kg(-1)), reaching high physiological plasma...... afferent nerves mediate sensory input from the gastrointestinal tract or pancreas; either directly or indirectly via the release of another mediator. GLP-2 receptors appear not be functionally expressed on vagal afferents....

  19. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Institute of Scientific and Technical Information of China (English)

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen


    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  20. A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression. (United States)

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Shende, Vikram; Dong, Bin; Liu, Jingwen


    It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP)D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3'UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation.

  1. The arginine vasopressin V1b receptor gene and prosociality: Mediation role of emotional empathy. (United States)

    Wu, Nan; Shang, Siyuan; Su, Yanjie


    The vasopressin V1b receptor (AVPR1B) gene has been shown to be closely associated with bipolar disorder and depression. However, whether it relates to positive social outcomes, such as empathy and prosocial behavior, remains unknown. This study explored the possible role of the AVPR1B gene rs28373064 in empathy and prosociality. A total of 256 men, who were genetically unrelated, non-clinical ethnic Han Chinese college students, participated in the study. Prosociality was tested by measuring the prosocial tendencies of cognitive and emotional empathy using the Interpersonal Reactivity Index (IRI). The single nucleotide polymorphism (SNP), rs28373064, was genotyped using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results suggest that the AVPR1B gene rs28373064 is linked to emotional empathy and prosociality. The mediation analysis indicated that the effect of the AVPR1B gene on prosociality might be mediated by emotional empathy. This study demonstrated the link between the AVPR1B gene and prosociality and provided evidence that emotional empathy might mediate the relation between the AVPR1B gene and prosociality.

  2. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement. (United States)

    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng


    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  3. Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth

    Institute of Scientific and Technical Information of China (English)

    Chen-Guang Bai; Xiao-Wei Hou; Feng Wang; Cen Qiu; Yan Zhu; Ling Huang; Jing Zhao


    AIM:To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth.METHODS:The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry,and the results were correlated with clinicopathological parameters,including the mitotic count,proliferative index (Ki-67 immunohistochemical staining),mitotic index (phospho-histone H3 immunohistochemical staining)and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling).Using primary cultured GIST cells,the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting,methyl thiazolyl tetrazolium (MTT),and apoptosis assays.RESULTS:We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples,respectively,and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis,including larger tumor size (P =0.0118),higher mitotic count (P =0.0058),higher proliferative index (P =0.0012),higher mitotic index (P =0.0282),lower apoptosis index (P =0.0484),and increased National Institutes of Health risk level (P =0.0012).We also found that the introduction of exogenous SCF potently increased KIT kinase activity,stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation,while a KIT immunoblocking antibody suppressed proliferation (P =0.01) and promoted apoptosis (P < 0.01)in cultured GIST cells.CONCLUSION:SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth.The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.

  4. Bradykinin and adenosine receptors mediate desflurane induced postconditioning in human myocardium: role of reactive oxygen species

    Directory of Open Access Journals (Sweden)

    Gérard Jean-Louis


    Full Text Available Abstract Background Desflurane during early reperfusion has been shown to postcondition human myocardium, in vitro. We investigated the role of adenosine and bradykinin receptors, and generation of radical oxygen species in desflurane-induced postconditioning in human myocardium. Methods We recorded isometric contraction of human right atrial trabeculae hanged in an oxygenated Tyrode's solution (34 degrees Celsius, stimulation frequency 1 Hz. After a 30-min hypoxic period, desflurane 6% was administered during the first 5 min of reoxygenation. Desflurane was administered alone or with pretreatment of N-mercaptopropionylglycine, a reactive oxygen species scavenger, 8-(p-Sulfophenyltheophylline, an adenosine receptor antagonist, HOE140, a selective B2 bradykinin receptor antagonist. In separate groups, adenosine and bradykinin were administered during the first minutes of reoxygenation alone or in presence of N-mercaptopropionylglycine. The force of contraction of trabeculae was recorded continuously. Developed force at the end of a 60-min reoxygenation period was compared (mean ± standard deviation between the groups by a variance analysis and post hoc test. Results Desflurane 6% (84 ± 6% of baseline enhanced the recovery of force after 60-min of reoxygenation as compared to control group (51 ± 8% of baseline, P N-mercaptopropionylglycine (54 ± 3% of baseline, 8-(p-Sulfophenyltheophylline (62 ± 9% of baseline, HOE140 (58 ± 6% of baseline abolished desflurane-induced postconditioning. Adenosine (80 ± 9% of baseline and bradykinin (83 ± 4% of baseline induced postconditioning (P vs control, N-mercaptopropionylglycine abolished the beneficial effects of adenosine and bradykinin (54 ± 8 and 58 ± 5% of baseline, respectively. Conclusions In vitro, desflurane-induced postconditioning depends on reactive oxygen species production, activation of adenosine and bradykinin B2 receptors. And, the cardioprotective effect of adenosine and bradykinin

  5. CB1 receptor mediates the effects of glucocorticoids on AMPK activity in the hypothalamus. (United States)

    Scerif, Miski; Füzesi, Tamás; Thomas, Julia D; Kola, Blerina; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta


    AMP-activated protein kinase (AMPK), a regulator of cellular and systemic energy homeostasis, can be influenced by several hormones. Tissue-specific alteration of AMPK activity by glucocorticoids may explain the increase in appetite, the accumulation of lipids in adipose tissues, and the detrimental cardiac effects of Cushing's syndrome. Endocannabinoids are known to mediate the effects of various hormones and to influence AMPK activity. Cannabinoids have central orexigenic and direct peripheral metabolic effects via the cannabinoid receptor type 1 (CB1). In our preliminary experiments, WT mice received implants of a corticosterone-containing pellet to establish a mouse model of Cushing's syndrome. Subsequently, WT and Cb1 (Cnr1)-knockout (CB1-KO) littermates were treated with corticosterone and AMPK activity in the hypothalamus, various adipose tissues, liver and cardiac tissue was measured. Corticosterone-treated CB1-KO mice showed a lack of weight gain and of increase in hypothalamic and hepatic AMPK activity. In adipose tissues, baseline AMPK activity was higher in CB1-KO mice, but a glucocorticoid-induced drop was observed, similar to that observed in WT mice. Cardiac AMPK levels were reduced in CB1-KO mice, but while WT mice showed significantly reduced AMPK activity following glucocorticoid treatment, CB1-KO mice showed a paradoxical increase. Our findings indicate the importance of the CB1 receptor in the central orexigenic effect of glucocorticoid-induced activation of hypothalamic AMPK activity. In the periphery adipose tissues, changes may occur independently of the CB1 receptor, but the receptor appears to alter the responsiveness of the liver and myocardial tissues to glucocorticoids. In conclusion, our data suggest that an intact cannabinoid pathway is required for the full metabolic effects of chronic glucocorticoid excess.

  6. Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of death receptor 5 of human colon cancer cells


    Mano Horinaka; Tatsushi Yoshida; Mitsuhiro Tomosugi; Shusuke Yasuda; Yoshihiro Sowa; Toshiyuki Sakai


    A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of death receptor 5 (DR5), a receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of...

  7. Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice


    Chia, Elizabeth; Kagota, Satomi; Wijekoon, Enoka P; McGuire, John J


    Background Under conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of re...

  8. mRNA expression of pattern recognition receptors and their signaling mediators in healthy and diseased gingival tissues



    Background: Gingivitis and periodontitis are initiated by inflammation caused by microorganisms. Pathogen-associated molecular patterns (PAMPs) from these microorganisms are recognized through various toll-like receptors (TLRs) and NOD-like receptors (NLRs). In this study, we have chosen five TLRs and two NLRs as representatives taking part in the recognition and inflammation process, along with a few of their signaling mediators including CD14, MYD88, and TRIF to compare their mRNA expressio...

  9. Endothelin potentiates TRPV1 via ETA receptor-mediated activation of protein kinase C

    Directory of Open Access Journals (Sweden)

    Furkert Jens


    Full Text Available Abstract Background Endothelin-1 (ET-1 both stimulates nociceptors and sensitizes them to noxious stimuli, an effect probably mediated by the ETA receptor (ETAR expressed in sensory neurons. The cellular mechanisms of this ET-1-mediated effect are only poorly understood. TRPV1, the heat-, pH- and capsaicin-sensitive cation channel already known to be modulated by a number of cellular mediators released in response to noxious stimuli and during inflammation, is a potential target for the action of ET-1. Results We studied the effects of ET-1 on TRPV1 in sensory neurons from the dorsal root ganglion (DRG and in HEK293 cells coexpressing TRPV1 and the ETAR. Specific 125I-ET-1 binding sites (817 ± 92 fmol/mg were detected in membrane preparations of DRG with an ETAR/ETBR ratio of 60:40. In an immunofluorescence analysis, coexpression of TRPV1 and the ETAR was found in a subpopulation of primary sensory neurons. ET-1 strongly potentiated capsaicin-induced TRPV1 currents in some neurons, and in HEK293 cells co-expressing TRPV1 and the ETAR. Weaker potentiation was observed in HEK293 cells coexpressing TRPV1 and the ETBR. ETAR activation also increased responses to low pH and heat. In HEK293 cells, strong potentiation of TRPV1 like that induced by ET-1 via the ETAR could be induced by PKC activation, but not with activators of the adenylyl cyclase or the PKA pathway. Furthermore, inhibition of PKC with bisindolylmaleimide X (BIM X or mutation of the PKC phosphorylation site S800 completely prevented ETAR-mediated potentiation. Conclusion We conclude that ET-1 potentiates TRPV1 by a PKC-dependent mechanism and that this could play a major role in the algogenic and hyperalgesic effects of ET-1 described in previous studies.

  10. Estrogen receptor-alpha mediates estrogen facilitation of baroreflex heart rate responses in conscious mice. (United States)

    Pamidimukkala, Jaya; Xue, Baojian; Newton, Leslie G; Lubahn, Dennis B; Hay, Meredith


    Estrogen facilitates baroreflex heart rate responses evoked by intravenous infusion of ANG II and phenylephrine (PE) in ovariectomized female mice. The present study aims to identify the estrogen receptor subtype involved in mediating these effects of estrogen. Baroreflex responses to PE, ANG II, and sodium nitroprusside (SNP) were tested in intact and ovariectomized estrogen receptor-alpha knockout (ERalphaKO) with (OvxE+) or without (OvxE-) estrogen replacement. Wild-type (WT) females homozygous for the ERalpha(+/+) were used as controls. Basal mean arterial pressures (MAP) and heart rates were comparable in all the groups except the ERalphaKO-OvxE+ mice. This group had significantly smaller resting MAP, suggesting an effect of estrogen on resting vascular tone possibly mediated by the ERbeta subtype. Unlike the WT females, estrogen did not facilitate baroreflex heart rate responses to either PE or ANG II in the ERalphaKO-OvxE+ mice. The slope of the line relating baroreflex heart rate decreases with increases in MAP evoked by PE was comparable in ERalphaKO-OvxE- (-6.97 +/- 1.4 beats.min(-1).mmHg(-1)) and ERalphaKO-OvxE+ (-6.18 +/- 1.3) mice. Likewise, the slope of the baroreflex bradycardic responses to ANG II was similar in ERalphaKO-OvxE- (-3.87 +/- 0.5) and ERalphaKO-OvxE+(-2.60 +/- 0.5) females. Data suggest that estrogen facilitation of baroreflex responses to PE and ANG II is predominantly mediated by ERalpha subtype. A second important observation in the present study is that the slope of ANG II-induced baroreflex bradycardia is significantly blunted compared with PE in the intact as well as the ERalphaKO-OvxE+ females. We have previously reported that this ANG II-mediated blunting of cardiac baroreflexes is observed only in WT males and not in ovariectomized WT females independent of their estrogen replacement status. The present data suggest that in females lacking ERalpha, ANG II causes blunting of cardiac baroreflexes similar to males and may be

  11. Autoimmunity in Arabidopsis acd11 Is Mediated by Epigenetic Regulation of an Immune Receptor

    DEFF Research Database (Denmark)

    Palma, K.; Thorgrimsen, S.; Malinovsky, F.G.;


    Certain pathogens deliver effectors into plant cells to modify host protein targets and thereby suppress immunity. These target modifications can be detected by intracellular immune receptors, or Resistance (R) proteins, that trigger strong immune responses including localized host cell death....... In a screen for lazarus (laz) mutants that suppress acd11 death we identified two genes, LAZ2 and LAZ5. LAZ2 encodes the histone lysine methyltransferase SDG8, previously shown to epigenetically regulate flowering time via modification of histone 3 (H3). LAZ5 encodes an RPS4-like R-protein, defined by several......, and that cell death in other lesion mimic mutants may also be caused by inappropriate activation of R genes. Moreover, SDG8 is required for basal and R protein-mediated pathogen resistance in Arabidopsis, revealing the importance of chromatin remodeling as a key process in plant innate immunity....

  12. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁


    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  13. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms (United States)

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.


    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  14. Src, a Molecular Switch Governing Gain Control of Synaptic Transmission Mediated by N-methyl-D-Aspartate Receptors (United States)

    Yu, Xian-Min; Salter, Michael W.


    The N-methyl-D-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

  15. Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis. (United States)

    Hammad, S M; Stefansson, S; Twal, W O; Drake, C J; Fleming, P; Remaley, A; Brewer, H B; Argraves, W S


    Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

  16. The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes. (United States)

    Fanger, N A; Cosman, D; Peterson, L; Braddy, S C; Maliszewski, C R; Borges, L


    The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.

  17. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium (United States)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.


    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  18. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling. (United States)

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N


    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  19. Orexin/hypocretin receptor 1 signaling mediates Pavlovian cue-food conditioning and extinction. (United States)

    Keefer, Sara E; Cole, Sindy; Petrovich, Gorica D


    Learned food cues can drive feeding in the absence of hunger, and orexin/hypocretin signaling is necessary for this type of overeating. The current study examined whether orexin also mediates cue-food learning during the acquisition and extinction of these associations. In Experiment 1, rats underwent two sessions of Pavlovian appetitive conditioning, consisting of tone-food presentations. Prior to each session, rats received either the orexin 1 receptor antagonist SB-334867 (SB) or vehicle systemically. SB treatment did not affect conditioned responses during the first conditioning session, measured as food cup behavior during the tone and latency to approach the food cup after the tone onset, compared to the vehicle group. During the second conditioning session, SB treatment attenuated learning. All groups that received SB, prior to either the first or second conditioning session, displayed significantly less food cup behavior and had longer latencies to approach the food cup after tone onset compared to the vehicle group. These findings suggest orexin signaling at the 1 receptor mediates the consolidation and recall of cue-food acquisition. In Experiment 2, another group of rats underwent tone-food conditioning sessions (drug free), followed by two extinction sessions under either SB or vehicle treatment. Similar to Experiment 1, SB did not affect conditioned responses during the first session. During the second extinction session, the group that received SB prior to the first extinction session, but vehicle prior to the second, expressed conditioned food cup responses longer after tone offset, when the pellets were previously delivered during conditioning, and maintained shorter latencies to approach the food cup compared to the other groups. The persistence of these conditioned behaviors indicates impairment in extinction consolidation due to SB treatment during the first extinction session. Together, these results demonstrate an important role for orexin

  20. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons. (United States)

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J


    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.

  1. Enhancement of potency and efficacy of NADA by PKC-mediated phosphorylation of vanilloid receptor. (United States)

    Premkumar, Louis S; Qi, Zhan-Heng; Van Buren, Jeremy; Raisinghani, Manish


    The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 microM NADA is 5-10% of the current induced by 1 microM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated ( approximately 15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (approximately 20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a approximately 15 mV depolarization and initiated a train of action potentials compared with 1 microM NADA that produced a approximately 5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.

  2. Endoglin-mediated suppression of prostate cancer invasion is regulated by activin and bone morphogenetic protein type II receptors.

    Directory of Open Access Journals (Sweden)

    Michael J Breen

    Full Text Available Mortality from prostate cancer (PCa is due to the formation of metastatic disease. Understanding how that process is regulated is therefore critical. We previously demonstrated that endoglin, a type III transforming growth factor β (TGFβ superfamily receptor, suppresses human PCa cell invasion and metastasis. Endoglin-mediated suppression of invasion was also shown by us to be dependent upon the type I TGFβ receptor, activin receptor-like kinase 2 (ALK2, and the downstream effector, Smad1. In this study we demonstrate for the first time that two type II TGFβ receptors are required for endoglin-mediated suppression of invasion: activin A receptor type IIA (ActRIIA and bone morphogenetic protein receptor type II (BMPRII. Downstream signaling through these receptors is predominantly mediated by Smad1. ActRIIA stimulates Smad1 activation in a kinase-dependent manner, and this is required for suppression of invasion. In contrast BMPRII regulates Smad1 in a biphasic manner, promoting Smad1 signaling through its kinase domain but suppressing it through its cytoplasmic tail. BMPRII's Smad1-regulatory effects are dependent upon its expression level. Further, its ability to suppress invasion is independent of either kinase function or tail domain. We demonstrate that ActRIIA and BMPRII physically interact, and that each also interacts with endoglin. The current findings demonstrate that both BMPRII and ActRIIA are necessary for endoglin-mediated suppression of human PCa cell invasion, that they have differential effects on Smad1 signaling, that they make separate contributions to regulation of invasion, and that they functionally and physically interact.

  3. Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons

    Directory of Open Access Journals (Sweden)

    Huang Li-Yen


    Full Text Available Abstract Prostaglandin E2 (PGE2 is a well-known inflammatory mediator that enhances the excitability of DRG neurons. Homomeric P2X3 and heteromeric P2X2/3 receptors are abundantly expressed in dorsal root ganglia (DRG neurons and participate in the transmission of nociceptive signals. The interaction between PGE2 and P2X3 receptors has not been well delineated. We studied the actions of PGE2 on ATP-activated currents in dissociated DRG neurons under voltage-clamp conditions. PGE2 had no effects on P2X2/3 receptor-mediated responses, but significantly potentiated fast-inactivating ATP currents mediated by homomeric P2X3 receptors. PGE2 exerted its action by activating EP3 receptors. To study the mechanism underlying the action of PGE2, we found that the adenylyl cyclase activator, forskolin and the membrane-permeable cAMP analogue, 8-Br-cAMP increased ATP currents, mimicking the effect of PGE2. In addition, forskolin occluded the enhancement produced by PGE2. The protein kinase A (PKA inhibitors, H89 and PKA-I blocked the PGE2 effect. In contrast, the PKC inhibitor, bisindolymaleimide (Bis did not change the potentiating action of PGE2. We further showed that PGE2 enhanced α,β-meATP-induced allodynia and hyperalgesia and the enhancement was blocked by H89. These observations suggest that PGE2 binds to EP3 receptors, resulting in the activation of cAMP/PKA signaling pathway and leading to an enhancement of P2X3 homomeric receptor-mediated ATP responses in DRG neurons.

  4. Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors. (United States)

    Dreiling, Michelle; Bischoff, Sabine; Schiffner, Rene; Rupprecht, Sven; Kiehntopf, Michael; Schubert, Harald; Witte, Otto W; Nathanielsz, Peter W; Schwab, Matthias; Rakers, Florian


    Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.

  5. Sucrose-induced analgesia in mice: Role of nitric oxide and opioid receptor-mediated system

    Directory of Open Access Journals (Sweden)

    Abtin Shahlaee


    Full Text Available Background: The mechanism of action of sweet substance-induced analgesia is thought to involve activation of the endogenous opioid system. The nitric oxide (NO pathway has a pivotal role in pain modulation of analgesic compounds such as opioids. Objectives: We investigated the role of NO and the opioid receptor-mediated system in the analgesic effect of sucrose ingestion in mice. Materials and Methods: We evaluated the effect of intraperitoneal administration of 10 mg/kg of NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME and 20 mg/kg of opioid receptor antagonist, naltrexone on the tail flick response in sucrose ingesting mice. Results: Sucrose ingestion for 12 days induced a statistically significant increase in the latency of tail flick response which was unmodified by L-NAME, but partially inhibited by naltrexone administration. Conclusions: Sucrose-induced nociception may be explained by facilitating the release of endogenous opioid peptides. Contrary to some previously studied pain models, the NO/cyclic guanosine monophosphate (cGMP pathway had no role in thermal hyperalgesia in our study. We recommend further studies on the involvement of NO in other animals and pain models.

  6. Pregnane X-receptor promotes stem cell-mediated colon cancer relapse. (United States)

    Planque, Chris; Rajabi, Fatemeh; Grillet, Fanny; Finetti, Pascal; Bertucci, François; Gironella, Meritxell; Lozano, Juan José; Beucher, Bertrand; Giraud, Julie; Garambois, Véronique; Vincent, Charles; Brown, Daniel; Caillo, Ludovic; Kantar, Jovana; Pelegrin, André; Prudhomme, Michel; Ripoche, Jérémie; Bourgaux, Jean François; Ginestier, Christophe; Castells, Antoni; Hollande, Frédéric; Pannequin, Julie; Pascussi, Jean Marc


    Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

  7. Metabotropic glutamate receptor-mediated long-term depression: molecular mechanisms. (United States)

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


    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.

  8. Pregnane X-receptor promotes stem cell-mediated colon cancer relapse (United States)

    Grillet, Fanny; Finetti, Pascal; Bertucci, François; Gironella, Meritxell; Lozano, Juan José; Beucher, Bertrand; Giraud, Julie; Garambois, Véronique; Vincent, Charles; Brown, Daniel; Caillo, Ludovic; Kantar, Jovana; Pelegrin, André; Prudhomme, Michel; Ripoche, Jérémie; Bourgaux, Jean François; Ginestier, Christophe; Castells, Antoni; Hollande, Frédéric; Pannequin, Julie; Pascussi, Jean Marc


    Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy. PMID:27448961

  9. Astrocytic β2-adrenergic receptors mediate hippocampal long-term memory consolidation

    KAUST Repository

    Gao, Virginia


    Emotionally relevant experiences form strong and long-lasting memories by critically engaging the stress hormone/neurotransmitter noradrenaline, which mediates and modulates the consolidation of these memories. Noradrenaline acts through adrenergic receptors (ARs), of which β2- Adrenergic receptors (βARs) are of particular importance. The differential anatomical and cellular distribution of βAR subtypes in the brain suggests that they play distinct roles in memory processing, although much about their specific contributions and mechanisms of action remains to be understood. Here we show that astrocytic rather than neuronal β2ARs in the hippocampus play a key role in the consolidation of a fear-based contextual memory. These hippocampal β2ARs, but not β1ARs, are coupled to the training-dependent release of lactate from astrocytes, which is necessary for long- Term memory formation and for underlying molecular changes. This key metabolic role of astrocytic β2ARs may represent a novel target mechanism for stress-related psychopathologies and neurodegeneration.

  10. Both neuropeptide Y knockdown and Y1 receptor inhibition modulate CART-mediated appetite control. (United States)

    Chu, Shu-Chen; Chen, Pei-Ni; Ho, Ying-Jui; Yu, Ching-Han; Hsieh, Yih-Shou; Kuo, Dong-Yih


    Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs.

  11. The cannabinoid receptor type 2 as mediator of mesenchymal stromal cell immunosuppressive properties.

    Directory of Open Access Journals (Sweden)

    Francesca Rossi

    Full Text Available Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians. Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes. In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells. We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.

  12. P2X7 receptor-mediated analgesia in cancer-induced bone pain

    DEFF Research Database (Denmark)

    Falk, Sarah; D. Schwab, Samantha; Frøsig-Jørgensen, Majbrit


    for cancer-induced bone pain. The P2X7 receptor (P2X7R) is involved in a variety of cellular functions and has been linked to both inflammatory and neuropathic pain. Here we study the analgesic potential of P2X7 receptor antagonism in a rat model of cancer-induced bone pain. In cancer-bearing animals, the P2......Pain is a common and debilitating complication for cancer patients significantly compromising their quality of life. Cancer-induced bone pain involves a complex interplay of molecular events, including mechanisms observed in inflammatory and neuropathic pain states, but also changes unique....... The results suggest that the P2X7R is involved in the mechanisms of cancer-induced bone pain, and that P2X7R antagonism might be a useful analgesic target. No effect was observed in sham or naïve animals, indicating that the P2X7R-mediated effect is state-dependent, and might therefore be an advantageous...

  13. Implications of astrocytes in mediating the protective effects of Selective Estrogen Receptor Modulators upon brain damage

    Directory of Open Access Journals (Sweden)

    George E. Barreto


    Full Text Available Selective Estrogen Receptor Modulators (SERMs are steroidal or non-steroidal compounds that are already used in clinical practice for the treatment of breast cancer, osteoporosis and menopausal symptoms. While SERMs actions in the breast, bone, and uterus have been well characterized, their actions in the brain are less well understood. Previous works have demonstrated the beneficial effects of SERMs in different chronic neurodegenerative diseases like Alzheimer, Parkinson’s disease and Multiple sclerosis, as well as acute degeneration as stroke and traumatic brain injury. Moreover, these compounds exhibit similar protective actions as those of estradiol in the Central Nervous System, overt any secondary effect. For these reasons, in the past few years, there has been a growing interest in the neuroprotective effects exerted directly or indirectly by SERMs in the SNC. In this context, astrocytes play an important role in the maintenance of brain metabolism, and antioxidant support to neurons, thus indicating that better protection of astrocytes are an important asset targeting neuronal protection. Moreover, various clinical and experimental studies have reported that astrocytes are essential for the neuroprotective effects of SERMs during neuronal injuries, as these cells express different estrogen receptors in cell membrane, demonstrating that part of SERMs effects upon injury may be mediated by astrocytes. The present work highlights the current evidence on the protective mechanisms of SERMs, such as tamoxifen and raloxifene, in the SNC, and their modulation of astrocytic properties as promising therapeutic targets during brain damage.

  14. Signaling mechanisms mediated by G-protein coupled receptors in human platelets

    Institute of Scientific and Technical Information of China (English)

    Sheikh Arshad SAEED; Huma RASHEED; Faisal A Wahed FECTO; Mohammad Ilyas ACHAKZAI; Rahmat ALI; John Dennis CONNOR; Anwar-ul-Hassan GILANI


    AIM: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA). METHODS: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 ℃ using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time. RESULTS: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1μmol/L) and AA (0.2μmol/L) was inhibited by the α2-receptor antagonist (yohimbine, IC50=0.6 μmol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC50=0.25 μmol/L).In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by calcium channel blockers, verapamil (IC50=0.4 μtmol/L) and diltiazem (IC50=2.5 μmol/L), as well as by low concentrations of inhibitors of phospholipase C (PLC) (U73122; IC50=0.2 μmol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC50=3.8 μmol/L). Herbimycin A, a specific inhibitor of tyrosine light chain kinase (TLCK), showed inhibition at IC50 value of 15 μmol/L, whereas chelerythrine, a protein kinase C (PKC)inhibitor, had no effect up to 20 μmol/L. CONCLUSION: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC,COX, and MAP kinase-signaling pathways.

  15. Prostaglandin receptor EP2 protects dopaminergic neurons against 6-OHDA-mediated low oxidative stress. (United States)

    Carrasco, Emilce; Werner, Peter; Casper, Diana


    Dopaminergic neurons in the substantia nigra (SN) selectively die in Parkinson's disease (PD), but it is unclear how and why this occurs. Recent findings implicate prostaglandin E(2) (PGE(2)) and two of its four receptors, namely EP1 and EP2, as mediators of degenerative and protective events in situations of acute and chronic neuronal death. EP1 activation can exacerbate excitotoxic damage in stroke models and our recent study showed that EP1 activation may explain the selective sensitivity of dopaminergic neurons to oxidative stress. Conversely, EP2 activation may be neuroprotective, although toxic effects have also been demonstrated. Here we investigated if and how EP2 activation might alter the survival of dopaminergic neurons following selective low-level oxidative injury evoked by the neurotoxin 6-hydroxydopamine (6-OHDA) in primary neuronal cultures prepared from embryonic rat midbrain. We found that cultured dopaminergic neurons displayed EP2 receptors. Butaprost, a selective EP2 agonist, significantly reduced 6-OHDA neurotoxicity. EP2 receptors are coupled to stimulatory G-proteins (Gs), which activate adenylate cyclase, increasing cAMP synthesis, which then activates protein kinase A (PKA). Both dibutyryl cAMP and forskolin reduced dopaminergic cell loss after 6-OHDA exposure. Conversely, KT5720 and H-89, two structurally distinct high-affinity PKA inhibitors, abolished the protective effect of butaprost, implicating cAMP-dependent PKA activity in the neuroprotection by EP2 activation. Finally, we show that melanized dopaminergic neurons in the human SN express EP2. This pathway warrants consideration as a neuroprotective strategy for PD.

  16. The membrane estrogen receptor GPR30 mediates cadmium-induced proliferation of breast cancer cells. (United States)

    Yu, Xinyuan; Filardo, Edward J; Shaikh, Zahir A


    Cadmium (Cd) is a nonessential metal that is dispersed throughout the environment. It is an endocrine-disrupting element which mimics estrogen, binds to estrogen receptor alpha (ERalpha), and promotes cell proliferation in breast cancer cells. We have previously published that Cd promotes activation of the extracellular regulated kinases, erk-1 and -2 in both ER-positive and ER-negative human breast cancer cells, suggesting that this estrogen-like effect of Cd is not associated with the ER. Here, we have investigated whether the newly appreciated transmembrane estrogen receptor, G-protein coupled receptor 30 (GPR30), may be involved in Cd-induced cell proliferation. Towards this end, we compared the effects of Cd in ER-negative human SKBR3 breast cancer cells in which endogenous GPR30 signaling was selectively inhibited using a GPR30 interfering mutant. We found that Cd concentrations from 50 to 500 nM induced a proliferative response in control vector-transfected SKBR3 cells but not in SKBR3 cells stably expressing interfering mutant. Similarly, intracellular cAMP levels increased about 2.4-fold in the vector transfectants but not in cells in which GPR30 was inactivated within 2.5 min after treatment with 500 nM Cd. Furthermore, Cd treatment rapidly activated (within 2.5 min) raf-1, mitogen-activated protein kinase kinase, mek-1, extracellular signal regulated kinases, erk-1/2, ribosomal S6 kinase, rsk, and E-26 like protein kinase, elk, about 4-fold in vector transfectants. In contrast, the activation of these signaling molecules in SKBR3 cells expressing the GPR30 mutant was only about 1.4-fold. These results demonstrate that Cd-induced breast cancer cell proliferation occurs through GPR30-mediated activation in a manner that is similar to that achieved by estrogen in these cells.

  17. Ionotropic glutamate receptors mediate inducible defense in the water flea Daphnia pulex.

    Directory of Open Access Journals (Sweden)

    Hitoshi Miyakawa

    Full Text Available Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean Daphnia pulex forms tooth-like defensive structures, "neckteeth," in response to chemical cues or signals, referred to as "kairomones," in this case released from phantom midge larvae, a predator of D. pulex. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in D. pulex, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in D. pulex. Moreover, using co-exposure of D. pulex to antagonists and juvenile hormone (JH, which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in D. pulex, but also will contribute to the understanding of complex mechanisms

  18. Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion. (United States)

    Rubí, Blanca; Ljubicic, Sanda; Pournourmohammadi, Shirin; Carobbio, Stefania; Armanet, Mathieu; Bartley, Clarissa; Maechler, Pierre


    Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 microM) and the D2-like receptor agonist quinpirole (5 microM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.

  19. Hindbrain GLP-1 receptor mediation of cisplatin-induced anorexia and nausea. (United States)

    De Jonghe, Bart C; Holland, Ruby A; Olivos, Diana R; Rupprecht, Laura E; Kanoski, Scott E; Hayes, Matthew R


    While chemotherapy-induced nausea and vomiting are clinically controlled in the acute (anorexia, nausea, fatigue, and other illness-type behaviors during the delayed phase (>24 h) of chemotherapy are largely uncontrolled. As the hindbrain glucagon-like peptide-1 (GLP-1) system contributes to energy balance and mediates aversive and stressful stimuli, here we examine the hypothesis that hindbrain GLP-1 signaling mediates aspects of chemotherapy-induced nausea and reductions in feeding behavior in rats. Specifically, hindbrain GLP-1 receptor (GLP-1R) blockade, via 4th intracerebroventricular (ICV) exendin-(9-39) injections, attenuates the anorexia, body weight reduction, and pica (nausea-induced ingestion of kaolin clay) elicited by cisplatin chemotherapy during the delayed phase (48 h) of chemotherapy-induced nausea. Additionally, the present data provide evidence that the central GLP-1-producing preproglucagon neurons in the nucleus tractus solitarius (NTS) of the caudal brainstem are activated by cisplatin during the delayed phase of chemotherapy-induced nausea, as cisplatin led to a significant increase in c-Fos immunoreactivity in NTS GLP-1-immunoreactive neurons. These data support a growing body of literature suggesting that the central GLP-1 system may be a potential pharmaceutical target for adjunct anti-emetics used to treat the delayed-phase of nausea and emesis, anorexia, and body weight loss that accompany chemotherapy treatments.

  20. AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke. (United States)

    Clarkson, Andrew N; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S Thomas


    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb control when administered after a delay from the stroke. Conversely, AMPAR antagonists impair motor recovery. The contributions of AMPARs to recovery are mediated by release of brain-derived neurotrophic factor (BDNF) in periinfarct cortex, as blocking local BDNF function in periinfarct cortex blocks AMPAR-mediated recovery and prevents the normal pattern of motor recovery. In contrast to a delayed AMPAR role in motor recovery, early administration of AMPAR agonists after stroke increases stroke damage. These findings indicate that the role of glutamate signaling through the AMPAR changes over time in stroke: early potentiation of AMPAR signaling worsens stroke damage, whereas later potentiation of the same signaling system improves functional recovery.

  1. Effects of environmental estrogenic chemicals on AP1 mediated transcription with estrogen receptors alpha and beta. (United States)

    Fujimoto, Nariaki; Honda, Hiroaki; Kitamura, Shigeyuki


    There has been much discussion concerning endocrine disrupting chemicals suspected of exerting adverse effects in both wildlife and humans. Since the majority of these compounds are estrogenic, a large number of in vitro tests for estrogenic characteristics have been developed for screening purpose. One reliable and widely used method is the reporter gene assay employing estrogen receptors (ERs) and a reporter gene with a cis-acting estrogen responsive element (ERE). Other elements such as AP1 also mediate estrogenic signals and the manner of response could be quite different from that of ERE. Since this has yet to be explored, the ER mediated AP1 activity in response to a series of environmental estrogens was investigated in comparison with ERE findings. All the compounds exhibited estrogenic properties with ERE-luc and their AP1 responses were quite similar. These was one exception, however, p,p'-DDT (1,1,1,-trichloro-2,2-bis(p-chlorophenyl)ethane) did not exert any AP1-luc activity, while it appeared to be estrogenic at 10(-7) to 10(-5)M with the ERE action. None of the compounds demonstrated ER beta:AP1 activity. These data suggest that significant differences can occur in responses through the two estrogen pathways depending on environmental chemicals.

  2. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis. (United States)

    Lionakis, Michail S; Fischer, Brett G; Lim, Jean K; Swamydas, Muthulekha; Wan, Wuzhou; Richard Lee, Chyi-Chia; Cohen, Jeffrey I; Scheinberg, Phillip; Gao, Ji-Liang; Murphy, Philip M


    Invasive candidiasis is the 4(th) leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo) to Ccr1(high) at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+) and Ccr1(-/-) donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+) recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+) cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

  3. Neonatal Fc receptor expression in dendritic cells mediates protective immunity against colorectal cancer. (United States)

    Baker, Kristi; Rath, Timo; Flak, Magdalena B; Arthur, Janelle C; Chen, Zhangguo; Glickman, Jonathan N; Zlobec, Inti; Karamitopoulou, Eva; Stachler, Matthew D; Odze, Robert D; Lencer, Wayne I; Jobin, Christian; Blumberg, Richard S


    Cancers arising in mucosal tissues account for a disproportionately large fraction of malignancies. Immunoglobulin G (IgG) and the neonatal Fc receptor for IgG (FcRn) have an important function in the mucosal immune system that we have now shown extends to the induction of CD8(+) T cell-mediated antitumor immunity. We demonstrate that FcRn within dendritic cells (DCs) was critical for homeostatic activation of mucosal CD8(+) T cells that drove protection against the development of colorectal cancers and lung metastases. FcRn-mediated tumor protection was driven by DCs activation of endogenous tumor-reactive CD8(+) T cells via the cross-presentation of IgG complexed antigens (IgG IC), as well as the induction of cytotoxicity-promoting cytokine secretion, particularly interleukin-12, both of which were independently triggered by the FcRn-IgG IC interaction in murine and human DCs. FcRn thus has a primary role within mucosal tissues in activating local immune responses that are critical for priming efficient anti-tumor immunosurveillance.

  4. Scavenger Receptor BI-mediated Selective Uptake Is Required for the Remodeling of High Density Lipoprotein by Endothelial Lipase

    NARCIS (Netherlands)

    Nijstad, Niels; Wiersma, Harmen; Gautier, Thomas; van der Giet, Markus; Maugeais, Cyrille; Tietge, Uwe J. F.


    Endothelial lipase (EL) is a negative regulator of high density lipoprotein (HDL) cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodeling of HDL. The present study investigates the requirement of SR-BI for the effects of EL- mediated phospholipid hydrolysis on HDL meta

  5. GLP-2 receptor localizes to enteric neurons and endocrine cells expressing vasoactive peptides and mediates increased blood flow

    DEFF Research Database (Denmark)

    Guan, Xinfu; Karpen, Heidi E; Stephens, John


    . These actions are mediated by the G-protein-coupled receptor, GLP-2R. Cellular localization of the GLP-2R and the nature of its signaling network in the gut, however, are poorly defined. Thus, our aim was to establish cellular localization of GLP-2R and functional connection to vascular action of GLP-2...

  6. Kinetics of the membrane current mediated by serotonin 5-HT3 receptors in cultured mouse neuroblastoma cells.

    NARCIS (Netherlands)

    Neijt, H.C.; Plomp, J.J.; Vijverberg, H.P.M.


    1. Ionic currents mediated by serotonin 5-HT3 receptors were studied in the mouse neuroblastoma cell line N1E-115, using suction pipettes for intracellular perfusion and voltage clamp recording. The dependence of the kinetics of the membrane current on serotonin concentration was investigated. 2. At

  7. Specific in vitro toxicity of crude and refined petroleum products. 1. Aryl hydrocarbon receptor-mediated responses.

    NARCIS (Netherlands)

    Vrabie, C.M.; Jonker, M.T.O.; Murk, A.J.


    The present study is the first in a series reporting on in vitro toxic potencies of oils. The objective was to determine whether 11 crude oils and refined products activate the aryl hydrocarbon receptor (AhR) in a dioxin receptor–mediated luciferase assay. Cells were exposed for 6 and 24 h to differ

  8. Histaminergic H1 receptors mediate L-histidine-induced anxiety in elevated plus-maze test in mice. (United States)

    Kumar, Kuchibhotla Vijaya; Krishna, Devarakonda Rama; Palit, Gautam


    The central histaminergic system is reported to mediate behavioural, hormonal and physiological homeostasis of living organisms. Recent reports indicate its prominent role in various neurobehavioural disorders such as depression and psychosis. This study evaluated the effect of activation of the central histaminergic system in anxiety-like conditions, using the elevated plus-maze test in mice, and elucidated the role of different histaminergic receptors mediating such effects. Peripheral administration of L-histidine (L-His), in a dose-dependent manner, significantly decreased the exploration time in open arms and number of entries into open arms without modifying the number of entries into closed arms of the elevated plus-maze, indicating anxiogenesis. Further, such effects of central histamine were significantly attenuated, in a dose-dependent manner, by pretreatment with pyrilamine (H1 receptor antagonist). Pretreatment with either zolantidine (H2 receptor antagonist) or thioperamide (H3 receptor antagonist), however, failed to attenuate the L-His-induced anxiogenesis. Our results indicate that anxiogenic effects of central histaminergic system appear to be mediated prominently by activation of H1 receptors.

  9. Critical role of interleukin-17/interleukin-17 receptor axis in mediating Con A-induced hepatitis. (United States)

    Yan, Shu; Wang, Luman; Liu, Nan; Wang, Ying; Chu, Yiwei


    Concanavalin A (Con A)-induced hepatitis is thought to be a T-cell-mediated disease with active destruction of liver cells. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells. However, whether IL-17/IL-17 receptor (IL-17/IL-17R)-mediated responses are involved in T-cell-mediated Con A-induced liver injury remains unclear. In this study, we found that IL-17 expression was highly elevated in liver tissues during Con A-induced hepatitis. The increased levels of IL-17 were paralleled with the severity of liver injury reflected by Alanine aminotransaminase and histological assay as well as the secretion of tumor necrosis factor (TNF)-α and IL-6. Blockage of IL-17 significantly ameliorated Con A-induced hepatitis, while overexpression of IL-17 systemically resulted in massive hepatocyte necrosis in mice. Furthermore, overexpression of an IL-17R immunoglobulin G1 fusion protein significantly attenuated liver inflammation after acute Con A treatment. High expression of IL-17R on Kupffer cells was also observed along with the production of cytokines including TNF-α and IL-6. Inhibition of Kupffer cells by gadolinium chloride completely prevented Con A-induced liver injury and cytokine release. Finally, IL-17-expressing CD4(+) T and natural killer T cells were greatly increased in Con A-injected mice compared with that in controls. Overall, our results indicate that IL-17R signaling is critically involved in the pathogenesis in Con A-induced hepatitis, and blockade of IL-17/IL-17R signaling pathway may represent a novel therapeutic intervention in human autoimmune-related hepatitis.

  10. NMDA receptors mediate stimulus-timing-dependent plasticity and neural synchrony in the dorsal cochlear nucleus

    Directory of Open Access Journals (Sweden)

    Roxana A Stefanescu


    Full Text Available Auditory information relayed by auditory nerve fibers and somatosensory information relayed by granule cell parallel fibers converge on the fusiform cells (FCs of the dorsal cochlear nucleus, the first brain station of the auditory pathway. In vitro, parallel fiber synapses on FCs exhibit spike-timing-dependent plasticity with Hebbian learning rules, partially mediated by the NMDA receptor (NMDAr. Well-timed bimodal auditory-somatosensory stimulation, in vivo equivalent of spike-timing-dependent plasticity, can induce stimulus-timing-dependent plasticity (StTDP of the FCs spontaneous and tone-evoked firing rates. In healthy guinea pigs, the resulting distribution of StTDP learning rules across a FC neural population is dominated by a Hebbian profile while anti-Hebbian, suppressive and enhancing LRs are less frequent. In this study, we investigate in vivo, the NMDAr contribution to FC baseline activity and long term plasticity. We find that blocking the NMDAr decreases the synchronization of FC- spontaneous activity and mediates differential modulation of FC rate-level functions such that low, and high threshold units are more likely to increase, and decrease, respectively, their maximum amplitudes. Three significant alterations in mean learning-rule profiles were identified: transitions from an initial Hebbian profile towards (1 an anti-Hebbian and (2 a suppressive profile, and (3 transitions from an anti-Hebbian to a Hebbian profile. FC units preserving their learning rules showed instead, NMDAr-dependent plasticity to unimodal acoustic stimulation, with persistent depression of tone-evoked responses changing to persistent enhancement following the NMDAr antagonist. These results reveal a crucial role of the NMDAr in mediating FC baseline activity and long-term plasticity which have important implications for signal processing and auditory pathologies related to maladaptive plasticity of dorsal cochlear nucleus circuitry.

  11. Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204)

    Energy Technology Data Exchange (ETDEWEB)

    Ohnishi, Koji; Komohara, Yoshihiro; Fujiwara, Yukio; Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Lei, XiaoFeng [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Nakagawa, Takenobu [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan); Department of Human Pathology, Institute of Health Biosciences, The University of Tokushima, Tokushima (Japan); Takeya, Motohiro, E-mail: [Department of Cell Pathology, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto (Japan)


    Highlights: {yields} We focused on the interaction between SR-A and TLR4 signaling in this study. {yields} SR-A deletion promoted NF{kappa}B activation in macrophages in septic model mouse. {yields} SR-A suppresses both MyD88-dependent and -independent TLR4 signaling in vitro. {yields} SR-A clears LPS binding to TLR4 which resulting in the suppression of TLR4 signals. -- Abstract: The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A{sup -/-}) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6 and interferon (IFN)-{beta} were significantly increased in SR-A{sup -/-} mice compared to wild-type mice, and elevated nuclear factor kappa B (NF{kappa}B) activation was detected in SR-A{sup -/-} macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NF{kappa}B in vitro. SR-A deletion also promoted the nuclear translocation of NF{kappa}B and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A{sup -/-} macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.

  12. Distinct muscarinic acetylcholine receptor subtypes mediate pre- and postsynaptic effects in rat neocortex

    Directory of Open Access Journals (Sweden)

    Gigout Sylvain


    Full Text Available Abstract Background Cholinergic transmission has been implicated in learning, memory and cognition. However, the cellular effects induced by muscarinic acetylcholine receptors (mAChRs activation are poorly understood in the neocortex. We investigated the effects of the cholinergic agonist carbachol (CCh and various agonists and antagonists on neuronal activity in rat neocortical slices using intracellular (sharp microelectrode and field potential recordings. Results CCh increased neuronal firing but reduced synaptic transmission. The increase of neuronal firing was antagonized by pirenzepine (M1/M4 mAChRs antagonist but not by AF-DX 116 (M2/M4 mAChRs antagonist. Pirenzepine reversed the depressant effect of CCh on excitatory postsynaptic potential (EPSP but had marginal effects when applied before CCh. AF-DX 116 antagonized the depression of EPSP when applied before or during CCh. CCh also decreased the paired-pulse inhibition of field potentials and the inhibitory conductances mediated by GABAA and GABAB receptors. The depression of paired-pulse inhibition was antagonized or prevented by AF-DX 116 or atropine but only marginally by pirenzepine. The inhibitory conductances were unaltered by xanomeline (M1/M4 mAChRs agonist, yet the CCh-induced depression was antagonized by AF-DX 116. Linopirdine, a selective M-current blocker, mimicked the effect of CCh on neuronal firing. However, linopirdine had no effect on the amplitude of EPSP or on the paired-pulse inhibition, indicating that M-current is involved in the increase of neuronal excitability but neither in the depression of EPSP nor paired-pulse inhibition. Conclusions These data indicate that the three effects are mediated by different mAChRs, the increase in firing being mediated by M1 mAChR, decrease of inhibition by M2 mAChR and depression of excitatory transmission by M4 mAChR. The depression of EPSP and increase of neuronal firing might enhance the signal-to-noise ratio, whereas the

  13. The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation. (United States)

    Oda, Yuko; Chalkley, Robert J; Burlingame, Alma L; Bikle, Daniel D


    Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation.

  14. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling (United States)

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.


    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

  15. A role of periaqueductal grey NR2B-containing NMDA receptor in mediating persistent inflammatory pain

    Directory of Open Access Journals (Sweden)

    Yang Qi


    Full Text Available Abstract The midbrain periaqueductal grey (PAG is a structure known for its roles in pain transmission and modulation. Noxious stimuli potentiate the glutamate synaptic transmission and enhance glutamate NMDA receptor expression in the PAG. However, little is known about roles of NMDA receptor subunits in the PAG in processing the persistent inflammatory pain. The present study was undertaken to investigate NR2A- and NR2B-containing NMDA receptors in the PAG and their modulation to the peripheral painful inflammation. Noxious stimuli induced by hind-paw injection of complete Freund's adjuvant (CFA caused up-regulation of NR2B-containing NMDA receptors in the PAG, while NR2A-containing NMDA receptors were not altered. Whole-cell patch-clamp recordings revealed that NMDA receptor mediated mEPSCs were increased significantly in the PAG synapse during the chronic phases of inflammatory pain in mice. PAG local infusion of Ro 25-6981, an NR2B antagonist, notably prolonged the paw withdrawal latency to thermal radian heat stimuli bilaterally in rats. Hyperoside (Hyp, one of the flavonoids compound isolated from Rhododendron ponticum L., significantly reversed up-regulation of NR2B-containing NMDA receptors in the PAG and exhibited analgesic activities against persistent inflammatory stimuli in mice. Our findings provide strong evidence that up-regulation of NR2B-containing NMDA receptors in the PAG involves in the modulation to the peripheral persistent inflammatory pain.

  16. Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation

    DEFF Research Database (Denmark)

    Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe


    The collagens of the extracellular matrix are the most abundant structural proteins in the mammalian body. In tissue remodeling and in the invasive growth of malignant tumors, collagens constitute an important barrier, and consequently, the turnover of collagen is a rate-limiting process...... in these events. A recently discovered turnover route with importance for tumor growth involves intracellular collagen degradation and is governed by the collagen receptor, urokinase plasminogen activator receptor-associated protein (uPARAP or Endo180). The interplay between this mechanism and extracellular...... collagenolysis is not known. In this report, we demonstrate the existence of a new, composite collagen breakdown pathway. Thus, fibroblast-mediated collagen degradation proceeds preferentially as a sequential mechanism in which extracellular collagenolysis is followed by uPARAP/Endo180-mediated endocytosis...

  17. Inhibitory nature of tiagabine-augmented GABAA receptor-mediated depolarizing responses in hippocampal pyramidal cells. (United States)

    Jackson, M F; Esplin, B; Capek, R


    . Independently of the stimulus intensity with which they were evoked, the charge transferred to the soma by excitatory postsynaptic currents (EPSCs), elicited in the presence of tiagabine (20 microM) during the large (1,428 +/- 331 pA) inward currents that underlie the DRs, was decreased on the average by 90.8 +/- 1.7%. Such inhibition occurred despite the presence of the GABAB receptor antagonist, CGP 52 432 (10 microM), indicating that GABAB heteroreceptors, located on glutamatergic terminals, do not mediate the observed reduction in the amplitude of excitatory postsynaptic responses. The present results suggest that despite facilitating the induction of GABA-mediated depolarizations, tiagabine application may nevertheless increase the effectiveness of synaptic inhibition during the synchronous high-frequency activation of inhibitory interneurons by enhanced shunting.

  18. Direct excitation of inhibitory interneurons by extracellular ATP mediated by P2Y1 receptors in the hippocampal slice. (United States)

    Kawamura, Masahito; Gachet, Christian; Inoue, Kazuhide; Kato, Fusao


    ATP is an important cell-to-cell signaling molecule mediating the interactions between astrocytes and neurons in the CNS. In the hippocampal slices, ATP suppresses excitatory transmission mostly through activation of adenosine A1 receptors, because the ectoenzyme activity for the extracellular breakdown of ATP to adenosine is high in slice preparations in contrast to culture environments. Because the hippocampus is also rich in the expression of P2 receptors activated specifically by ATP, we examined whether ATP modulates neuronal excitability in the acute slice preparations independently of adenosine receptors. Although ATP decreased the frequency of spontaneously occurring EPSCs in the CA3 pyramidal neurons through activation of adenosine A1 receptors, ATP concurrently increased the frequency of IPSCs in a manner dependent on action potential generation. This effect was mediated by P2Y1 receptors because (1) 2-methylthio-ATP (2meSATP) was the most potent agonist, (2) 2'-deoxy-N6-methyladenosine-3',5'-bisphosphate diammonium (MRS2179) abolished this effect, and (3) this increase in IPSC frequency was not observed in the transgenic mice lacking P2Y1 receptor proteins. Application of 2meSATP elicited MRS2179-sensitive time- and voltage-dependent inward currents in the interneurons, which depolarized the cell to firing threshold. Also, it increased [Ca2+]i in both astrocytes and interneurons, but, unlike the former effect, the latter was entirely dependent on Ca2+ entry. Thus, in hippocampal slices, in addition to activating A1 receptors of the excitatory terminals after being converted to adenosine, ATP activates P2Y1 receptors in the interneurons, which is linked to activation of unidentified excitatory conductance, through mechanisms distinct from those in the astrocytes.

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

    Directory of Open Access Journals (Sweden)

    Steven M Yellon

    Full Text Available 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.

  20. On the role of brain 5-HT7 receptor in the mechanism of hypothermia: comparison with hypothermia mediated via 5-HT1A and 5-HT3 receptor. (United States)

    Naumenko, Vladimir S; Kondaurova, Elena M; Popova, Nina K


    Intracerebroventricular administration of selective agonist of serotonin 5-HT(7) receptor LP44 (4-[2-(methylthio)phenyl]-N-(1,2,3,4-tetrahydro-1-naphthalenyl)-1-pyperasinehexanamide hydrochloride; 10.3, 20.5 or 41.0 nmol) produced considerable hypothermic response in CBA/Lac mice. LP44-induced (20.5 nmol) hypothermia was significantly attenuated by the selective 5-HT(7) receptor antagonist SB 269970 (16.1 fmol, i.c.v.) pretreatment. At the same time, intraperitoneal administration of LP44 in a wide range of doses 1.0, 2.0 or 10.0 mg/kg (2.0, 4.0, 20.0 μmol/kg) did not cause considerable hypothermic response. These findings indicate the implication of central, rather than peripheral 5-HT(7) receptors in the regulation of hypothermia. The comparison of LP44-induced (20.5 nmol) hypothermic reaction in eight inbred mouse strains (DBA/2J, CBA/Lac, C57BL/6, BALB/c, ICR, AKR/J, C3H and Asn) was performed and a significant effect of genotype was found. In the same eight mouse strains, functional activity of 5-HT(1A) and 5-HT(3) receptors was studied. The comparison of hypothermic responses produced by 5-HT(7) receptor agonist LP44 (20.5 nmol, i.c.v.) and 5-HT(1A) receptor agonist 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg), 5-HT(3) receptor agonist m-CPBG (40.0 nmol, i.c.v.) did not reveal considerable interstrain correlations between 5-HT(7) and 5-HT(1A) or 5-HT(3) receptor-induced hypothermia. The selective 5-HT(7) receptor antagonist SB 269970 (16.1 fmol, i.c.v.) failed to attenuate the hypothermic effect of 8-OH-DPAT 1.0 mg/kg, i.p. (3.0 μmol/kg) and m-CPBG (40.0 nmol, i.c.v.) indicating that the brain 5-HT(7) receptor is not involved in the hypothermic effects of 8-OH-DPAT or m-CPBG. The obtained results suggest that the central 5-HT(7) receptor plays an essential role in the mediation of thermoregulation independent of 5-HT(1A) and 5-HT(3) receptors.

  1. mu-opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2. (United States)

    Koch, Thomas; Seifert, Anja; Wu, Dai-Fei; Rankovic, Marija; Kraus, Jürgen; Börner, Christine; Brandenburg, Lars-Ove; Schröder, Helmut; Höllt, Volker


    We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

  2. Rapid effects of phytoestrogens on human colonic smooth muscle are mediated by oestrogen receptor beta.

    LENUS (Irish Health Repository)

    Hogan, A M


    Epidemiological studies have correlated consumption of dietary phytoestrogens with beneficial effects on colon, breast and prostate cancers. Genomic and non-genomic mechanisms are responsible for anti-carcinogenic effects but, until now, the effect on human colon was assumed to be passive and remote. No direct effect on human colonic smooth muscle has previously been described. Institutional research board approval was granted. Histologically normal colon was obtained from the proximal resection margin of colorectal carcinoma specimens. Circular smooth muscle strips were microdissected and suspended under 1g of tension in organ baths containing oxygenated Krebs solution at 37 degrees C. After an equilibration period, tissues were exposed to diarylpropionitrile (DPN) (ER beta agonist) and 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) (ER alpha agonist) or to the synthetic phytoestrogen compounds genistein (n=8), daidzein (n=8), fisetin (n=8) and quercetin (n=8) in the presence or absence of fulvestrant (oestrogen receptor antagonist). Mechanism of action was investigated by inhibition of downstream pathways. The cholinergic agonist carbachol was used to induce contractile activity. Tension was recorded isometrically. Phytoestrogens inhibit carbachol-induced colonic contractility. In keeping with a non-genomic, rapid onset direct action, the effect was within minutes, reversible and similar to previously described actions of 17 beta oestradiol. No effect was seen in the presence of fulvestrant indicating receptor modulation. While the DPN exerted inhibitory effects, PPT did not. The effect appears to be reliant on a p38\\/mitogen activated protein kinase mediated induction of nitric oxide production in colonic smooth muscle. The present data set provides the first description of a direct effect of genistein, daidzein, fisetin and quercetin on human colonic smooth muscle. The presence of ER in colonic smooth muscle has been functionally proven and the beta

  3. Beneficial effects of melatonin on in vitro bovine embryonic development are mediated by melatonin receptor 1. (United States)

    Wang, Feng; Tian, XiuZhi; Zhang, Lu; Gao, Chao; He, ChangJiu; Fu, Yao; Ji, PengYun; Li, Yu; Li, Ning; Liu, GuoShi


    In the current study, a fundamental question, that is, the mechanisms related to the beneficial effects of melatonin on mammalian embryonic development, was addressed. To examine the potential beneficial effects of melatonin on bovine embryonic development, different concentrations of melatonin (10(-11), 10(-9), 10(-7), 10(-5), 10(-3) M) were incubated with fertilized embryos. Melatonin in the range of 10(-11) to 10(-5) M significantly promoted embryonic development both in early culture medium (CR1aa +3 mg/mL BSA) and in later culture medium (CR1aa + 6%FBS). The most effective concentrations applied in the current studies were 10(-9) and 10(-7) M. Using quantitative real-time PCR with immunofluorescence and Western blot assays, the expression of melatonin receptor MT1 and MT2 genes was identified in bovine embryos. Further studies indicate that the beneficial effects of melatonin on bovine embryo development were mediated by the MT1 receptor. This is based on the facts that luzindole, a nonselective MT1 and MT2 antagonist, blocked the effect on melatonin-induced embryo development, while 4-P-PDOT, a selective MT2 antagonist, had little effect. Mechanistic explorations uncovered that melatonin application during bovine embryonic development significantly up-regulated the expression of antioxidative (Gpx4, SOD1, bcl-2) and developmentally important genes (SLC2A1, DNMT1A, and DSC2) while down-regulating expression of pro-apoptotic genes (P53, BAX, and Caspase-3). The results obtained from the current studies provide new information regarding the mechanisms by which melatonin promotes bovine embryonic development under both in vitro and in vivo conditions.

  4. Rapidly activated epidermal growth factor receptor mediates lipopolysaccharide-triggered migration of microglia. (United States)

    Qu, Wen-Sheng; Liu, Jun-Li; Li, Chun-Yu; Li, Xiao; Xie, Min-Jie; Wang, Wei; Tian, Dai-Shi


    Previous reports have suggested that epidermal growth factor receptor (EGFR) is involved in microglia activation characterized by cell morphology changes, cytokine production and cell migration; and the biochemical regulation of the microglia migration is a potential therapeutic target following CNS inflammatory damages. However, the role of EGFR in microglia motility after inflammatory stimulation remains unknown. In the present study, lipopolysaccharide (LPS) was found to trigger rapid EGFR phosphorylation within 10 min, which was sustained during long-term stimulation in both primary microglial cells and the cultured BV2 microglial cells, furthermore, blocking EGFR phosphorylation by AG1478 significantly attenuated the LPS-induced chemotactic and chemokinetic migration of microglia. In addition, LPS could initiate calcium oscillation in microglia during live-cell recording, however, an intracellular calcium chelator and a selective inhibitor of calcium/calmodulin-dependent protein kinase II, but not an extracellular calcium chelator, remarkably suppressed the LPS-induced EGFR phosphorylation in BV2 microglia cells. As EGFR is not a traditional receptor for LPS, these findings suggest that the rapid phosphorylation of EGFR is attributed to the LPS-triggered intracellular calcium mobilization. By examining the downstream signals of EGFR, we further proved that extracellular signal-regulated kinase (ERK) is essential for EGFR-mediated microglia migration, because ERK inhibition attenuated the chemotactic and chemokinetic migration of microglia that had been induced by either LPS or EGF. Collectively, these results suggest that LPS could trigger the rapid phosphorylation of EGFR and subsequent ERK activation through mobilizing calcium activity, which underlies the microglia migration in an inflammatory condition.

  5. Inhibition of monoacylglycerol lipase mediates a cannabinoid 1-receptor dependent delay of kindling progression in mice. (United States)

    von Rüden, E L; Bogdanovic, R M; Wotjak, C T; Potschka, H


    Endocannabinoids, including 2-arachidonoylglycerol (2-AG), activate presynaptic cannabinoid type 1 receptors (CB1R) on inhibitory and excitatory neurons, resulting in a decreased release of neurotransmitters. The event-specific activation of the endocannabinoid system by inhibition of the endocannabinoid degrading enzymes may offer a promising strategy to selectively activate CB1Rs at the site of excessive neuronal activation with the overall goal to prevent the development epilepsy. The aim of this study was to investigate the impact of monoacylglycerol lipase (MAGL) inhibition on the development and progression of epileptic seizures in the kindling model of temporal lobe epilepsy. Therefore, we selectively blocked MAGL by JZL184 (8mg/kg, i.p.) in mice to analyze the effects of increased 2-AG levels on kindling acquisition and to exclude an anticonvulsive potential. Our results showed that JZL184 treatment significantly delayed the development of generalized seizures (p=0.0066) and decreased seizure (pkindling model of temporal lobe epilepsy, but caused only modest effects in fully kindled mice. Moreover, we proved that JZL184 treatment had no effects in conditional CB1R knockout mice lacking expression of the receptor in principle neurons of the forebrain. In conclusion, the data demonstrate that indirect CB1R agonism delays the development of generalized epileptic seizures but has no relevant acute anticonvulsive effects. Furthermore, we confirmed that the effects of JZL184 on kindling progression are CB1R mediated. Thus, the data indicate that the endocannabinoid 2-AG might be a promising target for an anti-epileptogenic approach.

  6. Angiotensin II Type 1 Receptor-Mediated Electrical Remodeling in Mouse Cardiac Myocytes.

    Directory of Open Access Journals (Sweden)

    Jeremy Kim

    Full Text Available We recently characterized an autocrine renin angiotensin system (RAS in canine heart. Activation of Angiotensin II Type 1 Receptors (AT1Rs induced electrical remodeling, including inhibition of the transient outward potassium current Ito, prolongation of the action potential (AP, increased calcium entry and increased contractility. Electrical properties of the mouse heart are very different from those of dog heart, but if a similar system existed in mouse, it could be uniquely studied through genetic manipulations. To investigate the presence of a RAS in mouse, we measured APs and Ito in isolated myocytes. Application of angiotensin II (A2 for 2 or more hours reduced Ito magnitude, without affecting voltage dependence, and prolonged APs in a dose-dependent manner. Based on dose-inhibition curves, the fast and slow components of Ito (Ito,fast and IK,slow appeared to be coherently regulated by [A2], with 50% inhibition at an A2 concentration of about 400 nM. This very high K0.5 is inconsistent with systemic A2 effects, but is consistent with an autocrine RAS in mouse heart. Pre-application of the microtubule destabilizing agent colchicine eliminated A2 effects on Ito and AP duration, suggesting these effects depend on intracellular trafficking. Application of the biased agonist SII ([Sar1-Ile4-Ile8]A2, which stimulates receptor internalization without G protein activation, caused Ito reduction and AP prolongation similar to A2-induced changes. These data demonstrate AT1R mediated regulation of Ito in mouse heart. Moreover, all measured properties parallel those measured in dog heart, suggesting an autocrine RAS may be a fundamental feedback system that is present across species.

  7. P2X7 receptors mediate resistance to toxin-induced cell lysis. (United States)

    Schoenauer, Roman; Atanassoff, Alexander P; Wolfmeier, Heidi; Pelegrin, Pablo; Babiychuk, Eduard B; Draeger, Annette


    In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

  8. Minireview: osteoprotective action of estrogens is mediated by osteoclastic estrogen receptor-alpha. (United States)

    Imai, Yuuki; Kondoh, Shino; Kouzmenko, Alexander; Kato, Shigeaki


    The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-alpha or -beta or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERalpha, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERalpha through the shortened lifespan of osteoclasts.

  9. SIGIRR inhibits toll-like receptor 4, 5, 9-mediated immune responses in human airway epithelial cells. (United States)

    Zhang, Chun; Wu, Xueling; Zhao, Yunfeng; Deng, Zhaoxia; Qian, Guisheng


    Human airway epithelial cells (HAEC) may contribute to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) through toll-like receptors (TLRs)-mediated molecular mechanisms. TLRs exist on the surface of HAEC where binding to their cognate ligands initiates airway inflammation. Single immunoglobulin interleukin-1 receptor-related protein (SIGIRR) is a member of the toll-interleukin-1 receptor (TIR) family that can negatively modulate the immune response. We carried out studies to characterize SIGIRR modulation of TLR-mediated immune response in HAEC and to define its mechanisms of action. Following treatment with various concentrations of LPS, flagellin and CpG DNA, the levels of cognate TLRs 4, 5, and 9 were measured in the supernatants of HAEC over-expressing the SIGIRR molecule. Moreover, the interaction of the TLR adaptor myeloid differentiation factor 88 (MyD88) with SIGIRR in response to LPS-, flagellin- and CpG DNA-stimulation was examined by co-immunoprecipitation. The findings from this study revealed that overexpression of SIGIRR in HAEC stimulated by LPS, flagellin or CpG DNA resulted in attenuated production of the inflammatory mediators IL-6 and TNF-α. This attenuation was not the result of decreased expression of TLR4, 5 or 9, but rather a sequestration of MyD88 to the TLRs. In conclusion, SIGIRR can inhibit TLR4, 5, and 9-mediated immune responses in HAEC and may be a valuable therapeutic target for the prevention of ALI/ARDS.

  10. Methamphetamine-induced neurotoxicity and microglial activation are not mediated by fractalkine receptor signaling. (United States)

    Thomas, David M; Francescutti-Verbeem, Dina M; Kuhn, Donald M


    Methamphetamine (METH) damages dopamine (DA) nerve endings by a process that has been linked to microglial activation but the signaling pathways that mediate this response have not yet been delineated. Cardona et al. [Nat. Neurosci. 9 (2006), 917] recently identified the microglial-specific fractalkine receptor (CX3CR1) as an important mediator of MPTP-induced neurodegeneration of DA neurons. Because the CNS damage caused by METH and MPTP is highly selective for the DA neuronal system in mouse models of neurotoxicity, we hypothesized that the CX3CR1 plays a role in METH-induced neurotoxicity and microglial activation. Mice in which the CX3CR1 gene has been deleted and replaced with a cDNA encoding enhanced green fluorescent protein (eGFP) were treated with METH and examined for striatal neurotoxicity. METH depleted DA, caused microglial activation, and increased body temperature in CX3CR1 knockout mice to the same extent and over the same time course seen in wild-type controls. The effects of METH in CX3CR1 knockout mice were not gender-dependent and did not extend beyond the striatum. Striatal microglia expressing eGFP constitutively show morphological changes after METH that are characteristic of activation. This response was restricted to the striatum and contrasted sharply with unresponsive eGFP-microglia in surrounding brain areas that are not damaged by METH. We conclude from these studies that CX3CR1 signaling does not modulate METH neurotoxicity or microglial activation. Furthermore, it appears that striatal-resident microglia respond to METH with an activation cascade and then return to a surveying state without undergoing apoptosis or migration.

  11. Transient receptor potential ankyrin 1 mediates chronic pancreatitis pain in mice. (United States)

    Cattaruzza, Fiore; Johnson, Cali; Leggit, Alan; Grady, Eileen; Schenk, A Katrin; Cevikbas, Ferda; Cedron, Wendy; Bondada, Sandhya; Kirkwood, Rebekah; Malone, Brian; Steinhoff, Martin; Bunnett, Nigel; Kirkwood, Kimberly S


    Chronic pancreatitis (CP) is a devastating disease characterized by persistent and uncontrolled abdominal pain. Our lack of understanding is partially due to the lack of experimental models that mimic the human disease and also to the lack of validated behavioral measures of visceral pain. The ligand-gated cation channel transient receptor potential ankyrin 1 (TRPA1) mediates inflammation and pain in early experimental pancreatitis. It is unknown if TRPA1 causes fibrosis and sustained pancreatic pain. We induced CP by injecting the chemical agent trinitrobenzene sulfonic acid (TNBS), which causes severe acute pancreatitis, into the pancreatic duct of C57BL/6 trpa1(+/+) and trpa1(-/-) mice. Chronic inflammatory changes and pain behaviors were assessed after 2-3 wk. TNBS injection caused marked pancreatic fibrosis with increased collagen-staining intensity, atrophy, fatty replacement, monocyte infiltration, and pancreatic stellate cell activation, and these changes were reflected by increased histological damage scores. TNBS-injected animals showed mechanical hypersensitivity during von Frey filament probing of the abdomen, decreased daily voluntary wheel-running activity, and increased immobility scores during open-field testing. Pancreatic TNBS also reduced the threshold to hindpaw withdrawal to von Frey filament probing, suggesting central sensitization. Inflammatory changes and pain indexes were significantly reduced in trpa1(-/-) mice. In conclusion, we have characterized in mice a model of CP that resembles the human condition, with marked histological changes and behavioral measures of pain. We have demonstrated, using novel and objective pain measurements, that TRPA1 mediates inflammation and visceral hypersensitivity in CP and could be a therapeutic target for the treatment of sustained inflammatory abdominal pain.

  12. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases. (United States)

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P


    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases.

  13. Adolescent chronic mild stress alters hippocampal CB1 receptor-mediated excitatory neurotransmission and plasticity. (United States)

    Reich, C G; Mihalik, G R; Iskander, A N; Seckler, J C; Weiss, M S


    Endocannabinoids (eCBs) are involved in the stress response and alterations in eCB signaling may contribute to the etiology of mood disorders. Exposure to chronic mild stress (CMS), a model of depression, produces downregulation of the cannabinoid 1 (CB1) receptor in the hippocampus of male rats. However, it is unknown how this stress-induced change in CB1 levels affects eCB-mediated neurotransmission. In vitro, field potential recordings from CMS-exposed (21-days) rats were performed to assess the effects of stress on eCB-regulated glutamatergic neurotransmission in/on hippocampal area CA1. We observed that application of the CB1 agonist, WIN 55,212-5 (1 μM), in stress animals resulted in a ∼135% increase in excitatory neurotransmission, whereas CB1 activation in non-stress animals leads to a ∼30% decrease. However, during blockade of GABA(A) neurotransmission with picrotoxin, CB1 activation yielded a ∼35% decrease in stress animals. These findings indicate that CMS does not directly affect glutamatergic neurotransmission. Rather, CMS sensitizes CB1 function on GABAergic terminals, leading to less inhibition and an increase in excitatory neurotransmission. This finding is reinforced in that induction of weak long-term-potentiation (LTP) is enhanced in CMS-exposed animals compared to controls and this enhancement is CB1-dependent. Lastly, we observed that the LTP-blocking property of WIN 55,212-5 shifts from being glutamate-dependent in non-stress animals to being GABA-dependent in stress animals. These results effectively demonstrate that CMS significantly alters hippocampal eCB-mediated neurotransmission and synaptic plasticity.

  14. Androgen receptor is the key transcriptional mediator of the tumor suppressor SPOP in prostate cancer. (United States)

    Geng, Chuandong; Rajapakshe, Kimal; Shah, Shrijal S; Shou, John; Eedunuri, Vijay Kumar; Foley, Christopher; Fiskus, Warren; Rajendran, Mahitha; Chew, Sue Anne; Zimmermann, Martin; Bond, Richard; He, Bin; Coarfa, Cristian; Mitsiades, Nicholas


    Somatic missense mutations in the substrate-binding pocket of the E3 ubiquitin ligase adaptor SPOP are present in up to 15% of human prostate adenocarcinomas, but are rare in other malignancies, suggesting a prostate-specific mechanism of action. SPOP promotes ubiquitination and degradation of several protein substrates, including the androgen receptor (AR) coactivator SRC-3. However, the relative contributions that SPOP substrates may make to the pathophysiology of SPOP-mutant (mt) prostate adenocarcinomas are unknown. Using an unbiased bioinformatics approach, we determined that the gene expression profile of prostate adenocarcinoma cells engineered to express mt-SPOP overlaps greatly with the gene signature of both SRC-3 and AR transcriptional output, with a stronger similarity to AR than SRC-3. This finding suggests that in addition to its SRC-3-mediated effects, SPOP also exerts SRC-3-independent effects that are AR-mediated. Indeed, we found that wild-type (wt) but not prostate adenocarcinoma-associated mutants of SPOP promoted AR ubiquitination and degradation, acting directly through a SPOP-binding motif in the hinge region of AR. In support of these results, tumor xenografts composed of prostate adenocarcinoma cells expressing mt-SPOP exhibited higher AR protein levels and grew faster than tumors composed of prostate adenocarcinoma cells expressing wt-SPOP. Furthermore, genetic ablation of SPOP was sufficient to increase AR protein levels in mouse prostate. Examination of public human prostate adenocarcinoma datasets confirmed a strong link between transcriptomic profiles of mt-SPOP and AR. Overall, our studies highlight the AR axis as the key transcriptional output of SPOP in prostate adenocarcinoma and provide an explanation for the prostate-specific tumor suppressor role of wt-SPOP.

  15. Dab2, megalin, cubilin and amnionless receptor complex might mediate intestinal endocytosis in the suckling rat. (United States)

    Vázquez-Carretero, María D; Palomo, Marta; García-Miranda, Pablo; Sánchez-Aguayo, Inmaculada; Peral, María J; Calonge, María L; Ilundain, Anunciación A


    We previously proposed that Dab2 participates in the endocytosis of milk macromolecules in rat small intestine. Here we investigate the receptors that may mediate this endocytosis by studying the effects of age and diet on megalin, VLDLR, and ApoER2 expression, and that of age on the expression of cubilin and amnionless. Of megalin, VLDLR and ApoER2, only the megalin expression pattern resembles that of Dab2 previously reported. Thus the mRNA and protein levels of megalin and Dab2 are high in the intestine of the suckling rat, down-regulated by age and up-regulated by milk diet, mainly in the ileum. Neither age nor diet affect ApoER2 mRNA levels. The effect of age on VLDLR mRNA levels depends on the epithelial cell tested but they are down-regulated by milk diet. In the suckling rat, the intestinal expressions of both cubilin and amnionless are similar to that of megalin and megalin, cubilin, amnionless and Dab2 co-localize at the microvilli and in the apical endocytic apparatus. Co-localization of Dab2 with ApoER2 and VLDLR at the microvilli and in the apical endocytic apparatus is also observed. This is the first report showing intestinal co-localization of: megalin/cubilin/amnionless/Dab2, VLDLR/Dab2 and ApoER2/Dab2. We conclude that the megalin/cubilin/amnionless/Dab2 complex/es participate in intestinal processes, mainly during the lactation period and that Dab2 may act as an adaptor in intestinal processes mediated by ApoER2 and VLDLR.

  16. Age-dependent maturation of Toll-like receptor-mediated cytokine responses in Gambian infants.

    Directory of Open Access Journals (Sweden)

    Sarah Burl

    Full Text Available The global burden of neonatal and infant mortality due to infection is staggering, particularly in resource-poor settings. Early childhood vaccination is one of the major interventions that can reduce this burden, but there are specific limitations to inducing effective immunity in early life, including impaired neonatal leukocyte production of Th1-polarizing cytokines to many stimuli. Characterizing the ontogeny of Toll-like receptor (TLR-mediated innate immune responses in infants may shed light on susceptibility to infection in this vulnerable age group, and provide insights into TLR agonists as candidate adjuvants for improved neonatal vaccines. As little is known about the leukocyte responses of infants in resource-poor settings, we characterized production of Th1-, Th2-, and anti-inflammatory-cytokines in response to agonists of TLRs 1-9 in whole blood from 120 Gambian infants ranging from newborns (cord blood to 12 months of age. Most of the TLR agonists induced TNFα, IL-1β, IL-6, and IL-10 in cord blood. The greatest TNFα responses were observed for TLR4, -5, and -8 agonists, the highest being the thiazoloquinoline CLO75 (TLR7/8 that also uniquely induced cord blood IFNγ production. For most agonists, TLR-mediated TNFα and IFNγ responses increased from birth to 1 month of age. TLR8 agonists also induced the greatest production of the Th1-polarizing cytokines TNFα and IFNγ throughout the first year of life, although the relative responses to the single TLR8 agonist and the combined TLR7/8 agonist changed with age. In contrast, IL-1β, IL-6, and IL-10 responses to most agonists were robust at birth and remained stable through 12 months of age. These observations provide fresh insights into the ontogeny of innate immunity in African children, and may inform development of age-specific adjuvanted vaccine formulations important for global health.

  17. Spinal cord interneurons expressing the gastrin releasing peptide receptor convey itch through VGLUT2-mediated signaling. (United States)

    Aresh, Bejan; Freitag, Fabio B; Perry, Sharn; Blümel, Edda; Lau, Joey; Franck, Marina C M; Lagerström, Malin C


    Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR-population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in lamina II-IV. Application of the specific agonist gastrin releasing peptide (GRP) induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox;Grpr-Cre mice) displayed less spontaneous itch, and attenuated responses to both histaminergic and non-histaminergic agents. We could also show that application of the itch-inducing peptide natriuretic polypeptide b (NPPB) induces calcium influx in a sub-population of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

  18. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6. (United States)

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L; Byrne, Barbara A; Soulika, Athena M; Simon, Scott I; Isseroff, R Rivkah


    Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs.

  19. Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB₁ receptor blockade. (United States)

    Bellocchio, Luigi; Soria-Gómez, Edgar; Quarta, Carmelo; Metna-Laurent, Mathilde; Cardinal, Pierre; Binder, Elke; Cannich, Astrid; Delamarre, Anna; Häring, Martin; Martín-Fontecha, Mar; Vega, David; Leste-Lasserre, Thierry; Bartsch, Dusan; Monory, Krisztina; Lutz, Beat; Chaouloff, Francis; Pagotto, Uberto; Guzman, Manuel; Cota, Daniela; Marsicano, Giovanni


    Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of β-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral β-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors.

  20. Histamine H3 receptor activation prevents dopamine D1 receptor-mediated inhibition of dopamine release in the rat striatum: a microdialysis study. (United States)

    Alfaro-Rodriguez, Alfonso; Alonso-Spilsbury, María; Arch-Tirado, Emilio; Gonzalez-Pina, Rigoberto; Arias-Montaño, José-Antonio; Bueno-Nava, Antonio


    Histamine H3 receptors (H3Rs) co-localize with dopamine (DA) D1 receptors (D1Rs) on striatal medium spiny neurons and functionally antagonize D1R-mediated responses. The intra-striatal administration of D1R agonists reduces DA release whereas D1R antagonists have the opposite effect. In this work, a microdialysis method was used to study the effect of co-activating D1 and H3 receptors on the release of DA from the rat dorsal striatum. Infusion of the D1R agonist SKF-38393 (0.5 and 1 μM) significantly reduced DA release (26-58%), and this effect was prevented by co-administration of the H3R agonist immepip (10 μM). In turn, the effect of immepip was blocked by the H3R antagonist thioperamide (10 μM). Our results indicate that co-stimulation of post-synaptic D1 and H3 receptors may indirectly regulate basal DA release in the rat striatum and provide in vivo evidence for a functional interaction between D1 and H3 receptors in the basal ganglia.

  1. The intracellular juxtamembrane domain of discoidin domain receptor 2 (DDR2) is essential for receptor activation and DDR2-mediated cancer progression. (United States)

    Kim, Daehwan; Ko, Panseon; You, Eunae; Rhee, Sangmyung


    Discoidin domain receptors (DDRs) are unusual receptor tyrosine kinases (RTKs) that are activated by fibrillar collagens instead of soluble growth factors. DDRs play an important role in various cellular functions and disease processes, including malignant progression. Compared to other RTKs, DDRs have relatively long juxtamembrane domains, which are believed to contribute to receptor function. Despite this possibility, the function and mechanism of the juxtamembrane domain of DDRs have not yet been fully elucidated. In this study, we found that the cytoplasmic juxtamembrane 2 (JM2) region of DDR2 contributed to receptor dimerization, which is critical for receptor activation in response to collagen stimulation. A collagen-binding assay showed that JM2 was required for efficient binding of collagen to the discoidin (DS) domain. Immunohistochemical analysis of DDR2 expression using a tissue microarray demonstrated that DDR2 was overexpressed in several carcinoma tissues, including bladder, testis, lung, kidney, prostate and stomach. In H1299 cells, inhibition of DDR2 activity by overexpressing the juxtamembrane domain containing JM2 suppressed collagen-induced colony formation, cell proliferation and invasion via the inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9. Taken together, our results suggest that JM2-mediated dimerization is likely to be essential for DDR2 activation and cancer progression. Thus, inhibition of DDR2 function using a JM2-containing peptide might be a useful strategy for the treatment of DDR2-positive cancers.

  2. Α2 GABAA receptor sub-units in the ventral hippocampus and α5 GABAA receptor sub-units in the dorsal hippocampus mediate anxiety and fear memory. (United States)

    McEown, K; Treit, D


    Temporary neuronal inactivation of the ventral hippocampus with the GABAA agonist muscimol suppresses unconditioned fear behavior (anxiety) but inactivation of the dorsal hippocampus does not. On the other hand, inactivating the dorsal hippocampus disrupts fear memory, while inactivating the ventral hippocampus does not. Here we investigate the roles of hippocampal GABAA receptor sub-units in mediating these anxiolytic and amnesic effects of GABAA receptor agonists. We microinfused TPA023 (α2 agonist) or TB-21007 (inverse α5 agonist) into the dorsal or ventral hippocampus prior to testing rats in two animal models of anxiety: the elevated plus-maze and shock-probe burying test. Twenty-four hours later rats were re-tested in the shock-probe chamber with a non-electrified probe to assess their memory of the initial shock-probe experience (i.e., fear memory). We found that TPA023 was anxiolytic in the plus-maze and shock-probe burying tests when microinfused into the ventral hippocampus. However, TPA023 did not affect anxiety-related behavior when infused into the dorsal hippocampus. Conversely, we found that the α5 sub-unit inverse agonist TB-21007 impaired rats' memory of the initial shock-probe experience when infused into the dorsal hippocampus, but not when infused into the ventral hippocampus. This double dissociation suggests that α2 GABAA receptor sub-units in the ventral hippocampus mediate unconditioned fear or anxiety, while α5 GABAA receptor sub-units in the dorsal hippocampus mediate conditioned fear memory.

  3. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

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    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)


    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  4. Androgen receptor non-nuclear regulation of prostate cancer cell invasion mediated by Src and matriptase. (United States)

    Zarif, Jelani C; Lamb, Laura E; Schulz, Veronique V; Nollet, Eric A; Miranti, Cindy K


    Castration-resistant prostate cancers still depend on nuclear androgen receptor (AR) function despite their lack of dependence on exogenous androgen. Second generation anti-androgen therapies are more efficient at blocking nuclear AR; however resistant tumors still develop. Recent studies indicate Src is highly active in these resistant tumors. By manipulating AR activity in several different prostate cancer cell lines through RNAi, drug treatment, and the use of a nuclear-deficient AR mutant, we demonstrate that androgen acting on cytoplasmic AR rapidly stimulates Src tyrosine kinase via a non-genomic mechanism. Cytoplasmic AR, acting through Src enhances laminin integrin-dependent invasion. Active Matriptase, which cleaves laminin, is elevated within minutes after androgen stimulation, and is subsequently shed into the medium. Matriptase activation and shedding induced by cytoplasmic AR is dependent on Src. Concomitantly, CDCP1/gp140, a Matriptase and Src substrate that controls integrin-based migration, is activated. However, only inhibition of Matriptase, but not CDCP1, suppresses the AR/Src-dependent increase in invasion. Matriptase, present in conditioned medium from AR-stimulated cells, is sufficient to enhance invasion in the absence of androgen. Thus, invasion is stimulated by a rapid but sustained increase in Src activity, mediated non-genomically by cytoplasmic AR, leading to rapid activation and shedding of the laminin protease Matriptase.

  5. Evolutionary trace-based peptides identify a novel asymmetric interaction that mediates oligomerization in nuclear receptors. (United States)

    Gu, Peili; Morgan, Daniel H; Sattar, Minawar; Xu, Xueping; Wagner, Ryan; Raviscioni, Michele; Lichtarge, Olivier; Cooney, Austin J


    Germ cell nuclear factor (GCNF) is an orphan nuclear receptor that plays important roles in development and reproduction, by repressing the expression of essential genes such as Oct4, GDF9, and BMP15, through binding to DR0 elements. Surprisingly, whereas recombinant GCNF binds to DR0 sequences as a homodimer, endogenous GCNF does not exist as a homodimer but rather as part of a large complex termed the transiently retinoid-induced factor (TRIF). Here, we use evolutionary trace (ET) analysis to design mutations and peptides that probe the molecular basis for the formation of this unusual complex. We find that GCNF homodimerization and TRIF complex formation are DNA-dependent, and ET suggests that dimerization involves key functional sites on both helix 3 and helix 11, which are located on opposing surfaces of the ligand binding domain. Targeted mutations in either helix of GCNF disrupt the formation of both the homodimer and the endogenous TRIF complex. Moreover, peptide mimetics of both of these ET-determined sites inhibit dimerization and TRIF complex formation. This suggests that a novel helix 3-helix 11 heterotypic interaction mediates GCNF interaction and would facilitate oligomerization. Indeed, it was determined that the endogenous TRIF complex is composed of a GCNF oligomer. These findings shed light on an evolutionarily selected mechanism that reveals the unusual DNA-binding, dimerization, and oligomerization properties of GCNF.

  6. Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease (United States)

    Noelker, Carmen; Morel, Lydie; Lescot, Thomas; Osterloh, Anke; Alvarez-Fischer, Daniel; Breloer, Minka; Henze, Carmen; Depboylu, Candan; Skrzydelski, Delphine; Michel, Patrick P.; Dodel, Richard C.; Lu, Lixia; Hirsch, Etienne C.; Hunot, Stéphane; Hartmann, Andreas


    In mammalians, toll-like receptors (TLR) signal-transduction pathways induce the expression of a variety of immune-response genes, including inflammatory cytokines. It is therefore plausible to assume that TLRs are mediators in glial cells triggering the release of cytokines that ultimately kill DA neurons in the substantia nigra in Parkinson disease (PD). Accordingly, recent data indicate that TLR4 is up-regulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment in a mouse model of PD. Here, we wished to evaluate the role of TLR4 in the acute mouse MPTP model of PD: TLR4-deficient mice and wild-type littermates control mice were used for the acute administration way of MPTP or a corresponding volume of saline. We demonstrate that TLR4-deficient mice are less vulnerable to MPTP intoxication than wild-type mice and display a decreased number of Iba1+ and MHC II+ activated microglial cells after MPTP application, suggesting that the TLR4 pathway is involved in experimental PD. PMID:23462811

  7. Bladder cancer cell growth and motility implicate cannabinoid 2 receptor-mediated modifications of sphingolipids metabolism (United States)

    Bettiga, Arianna; Aureli, Massimo; Colciago, Giorgia; Murdica, Valentina; Moschini, Marco; Lucianò, Roberta; Canals, Daniel; Hannun, Yusuf; Hedlund, Petter; Lavorgna, Giovanni; Colombo, Renzo; Bassi, Rosaria; Samarani, Maura; Montorsi, Francesco; Salonia, Andrea; Benigni, Fabio


    The inhibitory effects demonstrated by activation of cannabinoid receptors (CB) on cancer proliferation and migration may also play critical roles in controlling bladder cancer (BC). CB expression on human normal and BC specimens was tested by immunohistochemistry. Human BC cells RT4 and RT112 were challenged with CB agonists and assessed for proliferation, apoptosis, and motility. Cellular sphingolipids (SL) constitution and metabolism were evaluated after metabolic labelling. CB1-2 were detected in BC specimens, but only CB2 was more expressed in the tumour. Both cell lines expressed similar CB2. Exposure to CB2 agonists inhibited BC growth, down-modulated Akt, induced caspase 3-activation and modified SL metabolism. Baseline SL analysis in cell lines showed differences linked to unique migratory behaviours and cytoskeletal re-arrangements. CB2 activation changed the SL composition of more aggressive RT112 cells by reducing (p < 0.01) Gb3 ganglioside (−50 ± 3%) and sphingosine 1-phosphate (S1P, −40 ± 4%), which ended up to reduction in cell motility (−46 ± 5%) with inhibition of p-SRC. CB2-selective antagonists, gene silencing and an inhibitor of SL biosynthesis partially prevented CB2 agonist-induced effects on cell viability and motility. CB2 activation led to ceramide-mediated BC cell apoptosis independently of SL constitutive composition, which instead was modulated by CB2 agonists to reduce cell motility. PMID:28191815

  8. Tumor suppressor SPOP mediates the proteasomal degradation of progesterone receptors (PRs) in breast cancer cells. (United States)

    Gao, Kun; Jin, Xiaofeng; Tang, Yan; Ma, Jian; Peng, Jingtiao; Yu, Long; Zhang, Pingzhao; Wang, Chenji


    Progesterone induces proliferation of breast cancer cells and contributes to the development of breast cancer. The effects of progesterone are mediated by progesterone receptors (PRs). However, it is still not fully understood how the proliferative effects of PR is regulated in vivo. Increasing amount of evidence strongly suggests that dysregulation of ubiquitin-proteasome system is closely associated with cancer pathogenesis. Speckle-type POZ protein (SPOP) is an adaptor protein of the CUL3-based E3 ubiquitin ligase complexes. SPOP represents one of the highest loci for loss of heterozygosity (LOH) in breast cancer. SPOP downregulation contributes to breast cancer cell growth and invasion. In this study, we revealed PR as a bona fide substrate for SPOP. SPOP interacts with PR in vivo and targets PR for ubiquitin-dependent proteasomal degradation. Moreover, SPOP suppresses progesteroneinduced PR transactivation, S phase entry, and Erk1/2 activation. Our study revealed novel molecular mechanisms underlying the regulation of PR protein homeostasis in breast cancer cells, and provided insights in understanding the relationship between SPOP inactivation and the development of breast cancer.

  9. Toll-Like Receptor 4 Engagement Mediates Prolyl Endopeptidase Release from Airway Epithelia via Exosomes. (United States)

    Szul, Tomasz; Bratcher, Preston E; Fraser, Kyle B; Kong, Michele; Tirouvanziam, Rabindra; Ingersoll, Sarah; Sztul, Elizabeth; Rangarajan, Sunil; Blalock, J Edwin; Xu, Xin; Gaggar, Amit


    Proteases are important regulators of pulmonary remodeling and airway inflammation. Recently, we have characterized the enzyme prolyl endopeptidase (PE), a serine peptidase, as a critical protease in the generation of the neutrophil chemoattractant tripeptide Pro-Gly-Pro (PGP) from collagen. However, PE has been characterized as a cytosolic enzyme, and the mechanism mediating PE release extracellularly remains unknown. We examined the role of exosomes derived from airway epithelia as a mechanism for PE release and the potential extracellular signals that regulate the release of these exosomes. We demonstrate a specific regulatory pathway of exosome release from airway epithelia and identify PE as novel exosome cargo. LPS stimulation of airway epithelial cells induces release of PE-containing exosomes, which is significantly attenuated by small interfering RNA depletion of Toll-like receptor 4 (TLR4). These differences were recapitulated upon intratracheal LPS administration in mice competent versus deficient for TLR4 signaling. Finally, sputum samples from subjects with cystic fibrosis colonized with Pseudomonas aeruginosa demonstrate elevated exosome content and increased PE levels. This TLR4-based mechanism highlights the first report of nonstochastic release of exosomes in the lung and couples TLR4 activation with matrikine generation. The increased quantity of these proteolytic exosomes in the airways of subjects with chronic lung disease highlights a new mechanism of injury and inflammation in the pathogenesis of pulmonary disorders.

  10. CCK1-Receptor Stimulation Protects Against Gut Mediator-Induced Lung Damage During Endotoxemia

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    Friederike Eisner


    Full Text Available Background/Aims: Cholecystokinin 1-receptor (CCK1-R activation by long chain fatty acid (LCFA absorption stimulates vago-vagal reflex pathways in the brain stem. The present study determines whether this reflex also activates the cholinergic anti-inflammatory pathway, a pathway known to modulate cytokine release during endotoxemia. Methods:Mesenteric lymph was obtained from wild type (WT and CCK1-R knockout (CCK1-R-/- mice intraperitoneally challenged with Lipopolysaccharid (LPS (endotoxemic lymph, EL and intestinally infused with vehicle or LCFA-enriched solution. The lymph was analyzed for TNFα, IL-6 and IL-10 concentration and administered to healthy recipient mice via jugular infusion. Alveolar wall thickness, myeloperoxidase (MPO and TUNEL positive cells were determined in lung tissue of recipient mice. Results: LCFA infusion in WT mice reduced TNFα concentration in EL by 49% compared to vehicle infusion, but had no effect in CCK1-R-/- mice. EL significantly increased the alveolar wall thickness, the number of MPO-positive and TUNEL-positive cells compared to control lymph administration. LCFA infusion in WT, but not in CCK1R-/- mice, significantly reduced these pathological effects of EL. Conclusion: During endotoxemia enteral LCFA absorption reduces TNFα release into mesenteric lymph and attenuates histomorphologic parameters of lung dysfunction. Failure to elicit this effect in CCK1R-/- mice demonstrates that anti-inflammatory properties of LCFAs are mediated through CCK1-Rs.

  11. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers. (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin


    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  12. The arcuate nucleus mediates GLP-1 receptor agonist liraglutide-dependent weight loss. (United States)

    Secher, Anna; Jelsing, Jacob; Baquero, Arian F; Hecksher-Sørensen, Jacob; Cowley, Michael A; Dalbøge, Louise S; Hansen, Gitte; Grove, Kevin L; Pyke, Charles; Raun, Kirsten; Schäffer, Lauge; Tang-Christensen, Mads; Verma, Saurabh; Witgen, Brent M; Vrang, Niels; Bjerre Knudsen, Lotte


    Liraglutide is a glucagon-like peptide-1 (GLP-1) analog marketed for the treatment of type 2 diabetes. Besides lowering blood glucose, liraglutide also reduces body weight. It is not fully understood how liraglutide induces weight loss or to what degree liraglutide acts directly in the brain. Here, we determined that liraglutide does not activate GLP-1-producing neurons in the hindbrain, and liraglutide-dependent body weight reduction in rats was independent of GLP-1 receptors (GLP-1Rs) in the vagus nerve, area postrema, and paraventricular nucleus. Peripheral injection of fluorescently labeled liraglutide in mice revealed the presence of the drug in the circumventricular organs. Moreover, labeled liraglutide bound neurons within the arcuate nucleus (ARC) and other discrete sites in the hypothalamus. GLP-1R was necessary for liraglutide uptake in the brain, as liraglutide binding was not seen in Glp1r(-/-) mice. In the ARC, liraglutide was internalized in neurons expressing proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART). Electrophysiological measurements of murine brain slices revealed that GLP-1 directly stimulates POMC/CART neurons and indirectly inhibits neurotransmission in neurons expressing neuropeptide Y (NPY) and agouti-related peptide (AgRP) via GABA-dependent signaling. Collectively, our findings indicate that the GLP-1R on POMC/CART-expressing ARC neurons likely mediates liraglutide-induced weight loss.

  13. γ-Glutamyltranspeptidase is an endogenous activator of Toll-like receptor 4-mediated osteoclastogenesis (United States)

    Moriwaki, Sawako; Into, Takeshi; Suzuki, Keiko; Miyauchi, Mutsumi; Takata, Takashi; Shibayama, Keigo; Niida, Shumpei


    Chronic inflammation-associated bone destruction, which is observed in rheumatoid arthritis (RA) and periodontitis, is mediated by excessive osteoclastogenesis. We showed previously that γ-glutamyltranspeptidase (GGT), an enzyme involved in glutathione metabolism, acts as an endogenous activator of such pathological osteoclastogenesis, independent of its enzymatic activity. GGT accumulation is clinically observed in the joints of RA patients, and, in animals, the administration of recombinant GGT to the gingival sulcus as an in vivo periodontitis model induces an increase in the number of osteoclasts. However, the underlying mechanisms of this process remain unclear. Here, we report that Toll-like receptor 4 (TLR4) recognizes GGT to activate inflammation-associated osteoclastogenesis. Unlike lipopolysaccharide, GGT is sensitive to proteinase K treatment and insensitive to polymyxin B treatment. TLR4 deficiency abrogates GGT-induced osteoclastogenesis and activation of NF-κB and MAPK signaling in precursor cells. Additionally, GGT does not induce osteoclastogenesis in cells lacking the signaling adaptor MyD88. The administration of GGT to the gingival sulcus induces increased osteoclastogenesis in wild-type mice, but does not induce it in TLR4-deficient mice. Our findings elucidate a novel mechanism of inflammation-associated osteoclastogenesis, which involves TLR4 recognition of GGT and subsequent activation of MyD88-dependent signaling. PMID:27775020

  14. Pax6 represses androgen receptor-mediated transactivation by inhibiting recruitment of the coactivator SPBP.

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    Julianne Elvenes

    Full Text Available The androgen receptor (AR has a central role in development and maintenance of the male reproductive system and in the etiology of prostate cancer. The transcription factor Pax6 has recently been reported to act as a repressor of AR and to be hypermethylated in prostate cancer cells. SPBP is a transcriptional regulator that previously has been shown to enhance the activity of Pax6. In this study we have identified SPBP to act as a transcriptional coactivator of AR. We also show that Pax6 inhibits SPBP-mediated enhancement of AR activity on the AR target gene probasin promoter, a repression that was partly reversed by increased expression of SPBP. Enhanced expression of Pax6 reduced the amount of SPBP associated with the probasin promoter when assayed by ChIP in HeLa cells. We mapped the interaction between both AR and SPBP, and AR and Pax6 to the DNA-binding domains of the involved proteins. Further binding studies revealed that Pax6 and SPBP compete for binding to AR. These results suggest that Pax6 represses AR activity by displacing and/or inhibiting recruitment of coactivators to AR target promoters. Understanding the mechanism for inhibition of AR coactivators can give rise to molecular targeted drugs for treatment of prostate cancer.

  15. N-methyl-D-aspartate receptor encephalitis mediates loss of intrinsic activity measured by functional MRI. (United States)

    Brier, Matthew R; Day, Gregory S; Snyder, Abraham Z; Tanenbaum, Aaron B; Ances, Beau M


    Spontaneous brain activity is required for the development and maintenance of normal brain function. Many disease processes disrupt the organization of intrinsic brain activity, but few pervasively reduce the amplitude of resting state blood oxygen level dependent (BOLD) fMRI fluctuations. We report the case of a female with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, longitudinally studied during the course of her illness to determine the contribution of NMDAR signaling to spontaneous brain activity. Resting state BOLD fMRI was measured at the height of her illness and 18 weeks following discharge from hospital. Conventional resting state networks were defined using established methods. Correlation and covariance matrices were calculated by extracting the BOLD time series from regions of interest and calculating either the correlation or covariance quantity. The intrinsic activity was compared between visits, and to expected activity from 45 similarly aged healthy individuals. Near the height of the illness, the patient exhibited profound loss of consciousness, high-amplitude slowing of the electroencephalogram, and a severe reduction in the amplitude of spontaneous BOLD fMRI fluctuations. The patient's neurological status and measures of intrinsic activity improved following treatment. We conclude that NMDAR-mediated signaling plays a critical role in the mechanisms that give rise to organized spontaneous brain activity. Loss of intrinsic activity is associated with profound disruptions of consciousness and cognition.

  16. Plasma extravasation mediated by lipopolysaccharide-induction of kinin B1 receptors in rat tissues

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    Paulo Roberto Wille


    Full Text Available The present study was performed to: (a evaluate the effects of kinin B1 (Sar{D-Phe8}-des-Arg9-BK; 10 nmol/kg and B2 (bradykinin (BK; 10 nmol/kg receptor agonists on plasma extravasation in selected rat tissues; (b determine the contribution of a lipopolysaccharide (LPS (100 μ g/kg to the effects triggered by B1 and B2 agonists; and (c characterize the selectivity of B1 ({Leu8}desArg9-BK; 10 nmol/kg and B2 (HOE 140; 10 nmol/kg antagonists as inhibitors of this kinin-induced phenomenon. B1 and B2 agonists were shown to increase plasma extravasation in the duodenum, ileum and also in the urinary bladder of the rat. LPS pretreatment enhanced the plasma extravasation mediated only by the B1 agonist in the duodenum, ileum, trachea, main and segmentar bronchi. These effects were prevented by the B1. but not the B2 antagonist. In normal rats, the B2 antagonist inhibited the effect of B2 agonist in all the tissues analyzed. However, in LPS-treated rats, the B2 antagonist was ineffective in the urinary bladder.

  17. Toll-like receptor 4 mediates the antitumor host response induced by Ganoderma atrum polysaccharide. (United States)

    Yu, Qiang; Nie, Shao-Ping; Wang, Jun-Qiao; Huang, Dan-Fei; Li, Wen-Juan; Xie, Ming-Yong


    The aim of this study is to investigate the role of Toll-like receptor (TLR) 4 in Ganoderma atrum polysaccharide (PSG-1)-induced antitumor activity. In vitro, the apoptosis rate of S-180 cells was increased in PSG-1-induced peritoneal macrophage derived from C3H/HeN (wild-type) mice, but not from C3H/HeJ (TLR4-deficient) mice. In the S-180 tumor model, phagocytosis, NO and ROS release, phosphorylation of MAPKs and Akt, and expression of NF-κB were increased by PSG-1 in peritoneal macrophage derived from C3H/HeN mice. Furthermore, PSG-1 elevated Th1 cytokine production and enhanced the cytotoxic activity of CTL and NK cells in C3H/HeN mice. In addition, PSG-1 decreased the tumor weight and increased the apoptosis rate and caspase-3 and caspase-9 activities of tumor derived from the C3H/HeN mice. However, none of these activities were observed in C3H/HeJ mice. In summary, these findings demonstrated that the antitumor activity of PSG-1 is mediated by TLR4.

  18. Gender-related differences in β-adrenergic receptor-mediated cardiac remodeling. (United States)

    Zhu, Baoling; Liu, Kai; Yang, Chengzhi; Qiao, Yuhui; Li, Zijian


    Cardiac remodeling is the pathological basis of various cardiovascular diseases. In this study, we found gender-related differences in β-adrenergic receptor (AR)-mediated pathological cardiac remodeling. Cardiac remodeling model was established by subcutaneous injection of isoprenaline (ISO) for 14 days. Heart rate (HR), mean arterial pressure (MAP), and echocardiography were obtained on 7th and 14th days during ISO administration. Myocardial cross-sectional area and the ratio of heart mass to tibia length (HM/TL) were detected to assess cardiac hypertrophy. Picro-Sirius red staining (picric acid + Sirius red F3B) was used to evaluate cardiac fibrosis. Myocardial capillary density was assessed by immunohistochemistry for von Willebrand factor. Further, real-time PCR was used to measure the expression of β1-AR and β2-AR. Results showed that ISO induced cardiac remodeling, the extent of which was different between female and male mice. The extent of increase in cardiac wall thickness, myocardial cross-sectional area, and collagen deposition in females was less than that in males. However, no gender-related difference was observed in HR, MAP, cardiac function, and myocardial capillary density. The distinctive decrease of β2-AR expression, rather than a decrease of β1-AR expression, seemed to result in gender-related differences in cardiac remodeling.

  19. Role of Fyn-mediated NMDA receptor function in prediabetic neuropathy in mice. (United States)

    Suo, Meng; Wang, Ping; Zhang, Mengyuan


    Diabetic neuropathy is a common complication of diabetes. This study evaluated the role of Fyn kinase and N-methyl-d-aspartate receptors (NMDARs) in the spinal cord in diabetic neuropathy using an animal model of high-fat diet-induced prediabetes. We found that prediabetic wild-type mice exhibited tactile allodynia and thermal hypoalgesia after a 16-wk high-fat diet, relative to normal diet-fed wild-type mice. Furthermore, prediabetic wild-type mice exhibited increased tactile allodynia and thermal hypoalgesia at 24 wk relative to 16 wk. Such phenomena were correlated with increased expression and activation of NR2B subunit of NMDARs, as well as Fyn-NR2B interaction in the spinal cord. Fyn(-/-) mice developed prediabetes after 16-wk high-fat diet treatment and exhibited thermal hypoalgesia, without showing tactile allodynia or altered expression and activation of NR2B subunit, relative to normal diet-fed Fyn(-/-) mice. Finally, intrathecal administrations of Ro 25-6981 (selective NR2B subunit-containing NMDAR antagonist) dose-dependently alleviated tactile allodynia, but not thermal hypoalgesia, at 16 and 24 wk in prediabetic wild-type mice. Our results suggested that Fyn-mediated NR2B signaling plays a critical role in regulation of prediabetic neuropathy and that the increased expression/function of NR2B subunit-containing NMDARs may contribute to the progression of neuropathy in type 2 diabetes.

  20. Estrogen controls vitamin D3-mediated resistance to experimental autoimmune encephalomyelitis by controlling vitamin D3 metabolism and receptor expression. (United States)

    Nashold, Faye E; Spach, Karen M; Spanier, Justin A; Hayes, Colleen E


    Multiple sclerosis (MS) is an autoimmune, neurodegenerative disease with a rapidly increasing female gender bias. MS prevalence decreases with increasing sunlight exposure, supporting our hypothesis that the sunlight-dependent hormone 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) is a natural inhibitor of autoimmune T cell responses in MS. We found that vitamin D(3) inhibited experimental autoimmune encephalomyelitis (EAE) in intact female mice, but not in ovariectomized females or males. To learn whether 17beta-estradiol (E(2)) is essential for vitamin D(3)-mediated protection, ovariectomized female mice were given E(2) or placebo and evaluated for vitamin D(3)-mediated EAE resistance. Diestrus-level E(2) implants alone provided no benefit, but they restored vitamin D(3)-mediated EAE resistance in the ovariectomized females. Synergy between E(2) and vitamin D(3) occurred through vitamin D(3)-mediated enhancement of E(2) synthesis, as well as E(2)-mediated enhancement of vitamin D receptor expression in the inflamed CNS. In males, E(2) implants did not enable vitamin D(3) to inhibit EAE. The finding that vitamin D(3)-mediated protection in EAE is female-specific and E(2)-dependent suggests that declining vitamin D(3) supplies due to sun avoidance might be contributing to the rapidly increasing female gender bias in MS. Moreover, declining E(2) synthesis and vitamin D(3)-mediated protection with increasing age might be contributing to MS disease progression in older women.

  1. Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor. (United States)

    Cherian, Priscilla P; Cheng, Benxu; Gu, Sumin; Sprague, Eugene; Bonewald, Lynda F; Jiang, Jean X


    Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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

  3. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes


    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.


    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  4. Endosome-mediated retrograde axonal transport of P2X3 receptor signals in primary sensory neurons

    Institute of Scientific and Technical Information of China (English)

    Xu-Qiao Chen; BinWang; Chengbiao Wu; Jin Pan; Bo Yuan; Yuan-Yuan Su; Xing-Yu Jiang; Xu Zhang; Lan Bao


    Neurotrophins and their receptors adopt signaling endosomes to transmit retrograde signals.However,the mechanisms of retrograde signaling for other ligand/receptor systems are poorly understood.Here,we report that the signals of the purinergic (P)2X3 receptor,an ATP-gated ion channel are retrogradely transported in dorsal root ganglion (DRG) neuron axons.We found that Rab5,a small GTPase,controls the early sorting of P2X3 receptors into endosomes,while Rab7 mediates the fast retrograde transport of P2X3 receptors.Intraplantar injection and axonal application into the microfluidic chamber of α,β-methylene-ATP (α,β-MeATP),a P2X selective agonist,enhanced the endocytosis and retrograde transport of P2X3 receptors.The α,β-MeATP-induced Ca2+ influx activated a pathway comprised of protein kinase C,rat sarcoma viral oncogene and extracellular signal-regulated protein kinase (ERK),which associated with endocytic P2X3 receptors to form signaling endosomes.Disruption of the lipid rafts abolished the α,β-MeATP-induced ERK phosphorylation,endocytosis and retrograde transport of P2X3 receptors.Furthermore,treatment of peripheral axons with α,β-MeATP increased the activation level of ERK and cAMP response element-binding protein in the cell bodies of DRG neurons and enhanced neuronal excitability.Impairment of either microtubule-based axonal transport in vivo or dynein function in vitro blocked α,β-MeATP-induced retrograde signals.These results indicate that P2X3 receptor-activated signals are transmitted via retrogradely transported endosomes in primary sensory neurons and provide a novel signaling mechanism for ligand-gated channels.

  5. Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi

    Directory of Open Access Journals (Sweden)

    Vikman Petter


    Full Text Available Abstract Background Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP from cigarette smoke, to nicotine and to DMSO, respectively. Methods Isolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ETB receptors and endothelin-1 (an ETA and ETB receptor agonist were studied by use of a sensitive myograph. Before ET-1 was introduced, the ETB receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ETA receptors. ETA and ETB receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ETA and ETB receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies. Results After being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ETA and ETB receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ETA and ETB receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ETA and ETB receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism. Conclusion The increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism.

  6. In adult female hamsters hypothyroidism stimulates D1 receptor-mediated breathing without altering D1 receptor expression. (United States)

    Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D


    Hypothyroidism affects cardiopulmonary regulation and function of dopaminergic receptors. Here we evaluated effects of 5 months of hypothyroidism on dopamine D1 receptor modulation of breathing in female hamsters using a D1 receptor antagonist SCH 23390. Euthyroid hamsters (EH) served as controls. Results indicated that hypothyroid female hamsters (HH) exhibited decreased body weights and minute ventilation (VE) following hypoxia due to decreased frequency of breathing (F). Moreover, SCH 23390 administration in HH increased VE by increasing tidal volume during exposure to air, hypoxia and following hypoxia. Relative to vehicle, SCH 23390 treatment decreased body temperature and hypoxic VE responsiveness in both groups. In EH, SCH 23390 decreased F in air, hypoxia and post hypoxia, and VE during hypoxia trended to decrease (P=0.053). Finally, expression of D1 receptor protein was not different between the two groups in any region evaluated. Thus, hypothyroidism in older female hamsters affected D1 receptor modulation of ventilation differently relative to euthyroid animals, but not expression of D1 receptors.

  7. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents


    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol


    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin-3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents....

  8. Pathogenesis of Aryl Hydrocarbon Receptor-Mediated Development of Lymphoma Is Associated with Increased Cyclooxygenase-2 Expression


    Vogel, Christoph F. A.; Li, Wen; Sciullo, Eric; Newman, John; Hammock, Bruce; Reader, J. Rachel; Tuscano, Joseph; Matsumura, Fumio


    Epidemiological studies indicate that exposure to environmental pollutants such as pesticides and dioxins leads to the pathogenesis of lymphoma and leukemia. Here, we show that activation of the aryl hydrocarbon receptor (AhR) by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in loss of the programmed cell death (apoptosis) response in three different lymphoma cell lines, which plays a key role in the development of cancer, especially lymphoma and leukemia. The AhR-mediated inhibition of...

  9. Pregnane X receptor (PXR-mediated gene repression and cross-talk of PXR with other nuclear receptors via coactivator interactions

    Directory of Open Access Journals (Sweden)

    Petr Pavek


    Full Text Available Pregnane X receptor is a ligand-activated nuclear receptor that mainly controls inducible expression of xenobiotics handling genes including biotransformation enzymes and drug transporters. Nowadays it is clear that PXR is also involved in regulation of intermediate metabolism through trans-activation and trans-repression of genes controlling glucose, lipid, cholesterol, bile acid and bilirubin homeostasis. In these processes PXR cross-talks with other nuclear receptors. Accumulating evidence suggests that the cross-talk is often mediated by competing for common coactivators or by disruption of coactivation and activity of other transcription factors by the ligand-activated PXR. In this respect mainly PXR-CAR and PXR-HNF4α interference have been reported and several cytochrome P450 enzymes (such as CYP7A1 and CYP8B1, phase II enzymes (SULT1E1, Gsta2, Ugt1a1, drug and endobiotic transporters (OCT1, Mrp2, Mrp3, Oatp1a and Oatp4 as well as intermediate metabolism enzymes (PEPCK1 and G6Pase have been shown as down-regulated genes after PXR activation. In this review, I summarize our current knowledge of PXR-mediated repression and coactivation interference in PXR-controlled gene expression regulation.

  10. Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL)

    DEFF Research Database (Denmark)

    Marques, Rita D; de Bruijn, Pauline I.A.; Sørensen, Mads Vaarby;


    Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors, which mediate significant inhibition of solute absorption. Recently, we identified several P2 receptors in the medullary thick ascending limb (m......TAL) including luminal and basolateral P2Y(2) receptors. In addition, we found evidence for a basolateral P2X receptor. Here we investigate the effect of basolateral ATP on NaCl absorption in isolated, perfused mouse mTALs using the electrical measurement of equivalent short circuit current (I'(sc)). Non...... receptor antagonist suramin blocked the effect. P2Y receptors were found not to be involved in this effect. The P2X receptor agonist 2MeSATP mimicked the ATP effect and the P2X receptor antagonist oATP blocked it. In P2X(7)(-/-) mice the ATP effect remained unaltered. In contrast, in P2X(4)(-/-) mice...

  11. Tyrosine-based signal mediates LRP6 receptor endocytosis and desensitization of Wnt/β-catenin pathway signaling. (United States)

    Liu, Chia-Chen; Kanekiyo, Takahisa; Roth, Barbara; Bu, Guojun


    Wnt/β-catenin signaling orchestrates a number of critical events including cell growth, differentiation, and cell survival during development. Misregulation of this pathway leads to various human diseases, specifically cancers. Endocytosis and phosphorylation of the LDL receptor-related protein 6 (LRP6), an essential co-receptor for Wnt/β-catenin signaling, play a vital role in mediating Wnt/β-catenin signal transduction. However, its regulatory mechanism is not fully understood. In this study, we define the mechanisms by which LRP6 endocytic trafficking regulates Wnt/β-catenin signaling activation. We show that LRP6 mutant with defective tyrosine-based signal in its cytoplasmic tail has an increased cell surface distribution and decreased endocytosis rate. These changes in LRP6 endocytosis coincide with an increased distribution to caveolae, increased phosphorylation, and enhanced Wnt/β-catenin signaling. We further demonstrate that treatment of Wnt3a ligands or blocking the clathrin-mediated endocytosis of LRP6 leads to a redistribution of wild-type receptor to lipid rafts. The LRP6 tyrosine mutant also exhibited an increase in signaling activation in response to Wnt3a stimulation when compared with wild-type LRP6, and this activation is suppressed when caveolae-mediated endocytosis is blocked. Our results reveal molecular mechanisms by which LRP6 endocytosis routes regulate its phosphorylation and the strength of Wnt/β-catenin signaling, and have implications on how this pathway can be modulated in human diseases.

  12. Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors. (United States)

    Guo, Shuohan; Zhang, Xiaohan; Zheng, Mei; Zhang, Xiaowei; Min, Chengchun; Wang, Zengtao; Cheon, Seung Hoon; Oak, Min-Ho; Nah, Seung-Yeol; Kim, Kyeong-Man


    Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the β2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of β2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

  13. NR4A nuclear receptors mediate carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600

    Energy Technology Data Exchange (ETDEWEB)

    Ishizawa, Michiyasu [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan); Kagechika, Hiroyuki [Graduate School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062 (Japan); Makishima, Makoto, E-mail: [Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610 (Japan)


    Highlights: Black-Right-Pointing-Pointer The function of RXR heterodimers with NR4 receptors remains unknown. Black-Right-Pointing-Pointer The RXR ligand HX600 induces expression of carnitine palmitoyltransferase 1A (CPT1A). Black-Right-Pointing-Pointer HX600-induced CPT1A expression is mediated by the NR4 receptors, Nur77 and NURR1. Black-Right-Pointing-Pointer CPT1A induction by HX600 is not mediated by de novo protein synthesis. Black-Right-Pointing-Pointer CPT1A could be a target of the Nur77-RXR and NURR1-RXR heterodimers. -- Abstract: Retinoid X receptors (RXRs) are members of the nuclear receptor superfamily and can be activated by 9-cis retinoic acid (9CRA). RXRs form homodimers and heterodimers with other nuclear receptors such as the retinoic acid receptor and NR4 subfamily nuclear receptors, Nur77 and NURR1. Potential physiological roles of the Nur77-RXR and NURR1-RXR heterodimers have not been elucidated. In this study, we identified a gene regulated by these heterodimers utilizing HX600, a selective RXR agonist for Nur77-RXR and NURR1-RXR. While 9CRA induced many genes, including RAR-target genes, HX600 effectively induced only carnitine palmitoyltransferase 1A (CPT1A) in human teratocarcinoma NT2/D1 cells, which express RXR{alpha}, Nur77 and NURR1. HX600 also increased CPT1A expression in human embryonic kidney (HEK) 293 cells and hepatocyte-derived HepG2 cells. Although HX600 induced CPT1A less effectively than 9CRA, overexpression of Nur77 or NURR1 increased the HX600 response to levels similar to 9CRA in NT2/D1 and HEK293 cells. A dominant-negative form of Nur77 or NURR1 repressed the induction of CPT1A by HX600. A protein synthesis inhibitor did not alter HX600-dependent CPT1A induction. Thus, the rexinoid HX600 directly induces expression of CPT1A through a Nur77 or NURR1-mediated mechanism. CPT1A, a gene involved in fatty acid {beta}-oxidation, could be a target of RXR-NR4 receptor heterodimers.

  14. Cocaine exposure impairs multilineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor [corrected]. (United States)

    Nixon, Christopher C; Schwartz, Brandon H; Dixit, Dhaval; Zack, Jerome A; Vatakis, Dimitrios N


    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade.

  15. Down-regulation of the A3 adenosine receptor in human mast cells upregulates mediators of angiogenesis and remodeling. (United States)

    Rudich, Noam; Dekel, Ornit; Sagi-Eisenberg, Ronit


    Adenosine activated mast cells have been long implicated in allergic asthma and studies in rodent mast cells have assigned the A3 adenosine receptor (A3R) a primary role in mediating adenosine responses. Here we analyzed the functional impact of A3R activation on genes that are implicated in tissue remodeling in severe asthma in the human mast cell line HMC-1 that shares similarities with lung derived human mast cells. Quantitative real time PCR demonstrated upregulation of IL6, IL8, VEGF, amphiregulin and osteopontin. Moreover, further upregulation of these genes was noted upon the addition of dexamethasone. Unexpectedly, activated A3R down regulated its own expression and knockdown of the receptor replicated the pattern of agonist induced gene upregulation. This study therefore identifies the human mast cell A3R as regulator of tissue remodeling gene expression in human mast cells and demonstrates a heretofore-unrecognized mode of feedback regulation that is exerted by this receptor.

  16. A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development. (United States)

    Kitayama, Midori; Mizutani, Kiyohito; Maruoka, Masahiro; Mandai, Kenji; Sakakibara, Shotaro; Ueda, Yuki; Komori, Takahide; Shimono, Yohei; Takai, Yoshimi


    Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.

  17. Suppression of sustained and transient ON signals of amacrine cells by GABA is mediated by different receptor subtypes

    Institute of Scientific and Technical Information of China (English)

    张道启; 杨如; 杨雄里


    Intracellular recordings were made from amacrine cells in the isolated, superfused carp retina, and the effects of γ-aminobutyric acid (GABA) on sustained and transient ON signals of these cells were studied. Exogenous GABA application partially suppressed the sustained response of ON amacrine cells, which could be completely reversed by picrotoxin (PTX), a chloride channel blocker, and by bicuculline (BCC), a specific GABA_A receptor antagonist. On the other hand, suppression by GABA of the ON response which was predominantly driven by rod signals in a certain portion of transient ON-OFF amacrine cells was completely blocked by PTX, but not by BCC, indicating that GABA_C receptors may be involved in the effect. These results suggest that GABA_A and GABA_C receptors may be respectively involved in mediating the transmission of sustained and transient signals in the carp inner retina.

  18. Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa (United States)

    Menegazzo, Massimo; Magagna, Sabina; Di Nisio, Andrea; Šabović, Iva; Rocca, Maria Santa; Scattolini, Valentina; Filippi, Andrea; Foresta, Carlo


    The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31–37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis. PMID:27992447

  19. Receptor-Mediated Surface Charge Inversion Platform Based on Porous Silicon Nanoparticles for Efficient Cancer Cell Recognition and Combination Therapy. (United States)

    Zhang, Feng; Correia, Alexandra; Mäkilä, Ermei; Li, Wei; Salonen, Jarno; Hirvonen, Jouni J; Zhang, Hongbo; Santos, Hélder A


    Negatively charged surface-modified drug delivery systems are promising for in vivo applications as they have more tendency to accumulate in tumor tissues. However, the inefficient cell uptake of these systems restricts their final therapeutic performance. Here, we have fabricated a receptor-mediated surface charge inversion nanoparticle made of undecylenic acid modified, thermally hydrocarbonized porous silicon (UnTHCPSi) nanoparticles core and sequentially modified with polyethylenimine (PEI), methotrexate (MTX), and DNA aptamer AS1411 (herein termed as UnTHCPSi-PEI-MTX@AS1411) for enhancing the cell uptake of nucleolin-positive cells. The efficient interaction of AS1411 and the relevant receptor nucleolin caused the disintegration of the negative-charged AS1411 surface. The subsequent surface charge inversion and exposure of the active targeting ligand, MTX, enhanced the cell uptake of the nanoparticles. On the basis of this synergistic effect, the UnTHCPSi-PEI-MTX@AS1411 (hydrodynamic diameter is 242 nm) were efficiently internalized by nucleolin-positive MDA-MB-231 breast cancer cells, with an efficiency around 5.8 times higher than that of nucleolin-negative cells (NIH 3T3 fibroblasts). The receptor competition assay demonstrated that the major mechanism (more than one-half) of the internalized nanoparticles in MDA-MB-231 cells was due to the receptor-mediated surface charge inversion process. Finally, after loading of sorafenib, the nanosystem showed efficient performance for combination therapy with an inhibition ratio of 35.6%.

  20. Folate-receptor-mediated delivery of InP quantum dots for bioimaging using confocal and two-photon microscopy. (United States)

    Bharali, Dhruba J; Lucey, Derrick W; Jayakumar, Harishankar; Pudavar, Haridas E; Prasad, Paras N


    A novel method for the synthesis of highly monodispersed hydrophillic InP-ZnS nanocrystals and their use as luminescence probes for live cell imaging is reported. Hydrophobic InP-ZnS nanocrystals are prepared by a new method that yields high-quality, luminescent core-shell nanocrystals within 6-8 h of total reaction time. Then by carefully manipulating the surface of these passivated nanocrystals, aqueous dispersions of folate-conjugated nanocrystals (folate-QDs) with high photostability are prepared. By use of confocal microscopy, we demonstrate the receptor-mediated delivery of folic acid conjugated quantum dots into folate-receptor-positive cell lines such as KB cells. These folate-QDs tend to accumulate in multi-vescicular bodies of KB cells after 6 h of incubation. Receptor-mediated delivery was confirmed by comparison with the uptake of these particles in folate-receptor-negative cell lines such as A549. Efficient two-photon excitation of these particles and two-photon imaging using these particles are also demonstrated. The use of these InP-ZnS nanoparticles and their efficient two-photon excitation can be potentially useful for deep tissue imaging for future in vivo studies.

  1. A novel ecdysone receptor mediates steroid-regulated developmental events during the mid-third instar of Drosophila.

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    Benjamin F B Costantino


    Full Text Available The larval salivary gland of Drosophila melanogaster synthesizes and secretes glue glycoproteins that cement developing animals to a solid surface during metamorphosis. The steroid hormone 20-hydroxyecdysone (20E is an essential signaling molecule that modulates most of the physiological functions of the larval gland. At the end of larval development, it is known that 20E--signaling through a nuclear receptor heterodimer consisting of EcR and USP--induces the early and late puffing cascade of the polytene chromosomes and causes the exocytosis of stored glue granules into the lumen of the gland. It has also been reported that an earlier pulse of hormone induces the temporally and spatially specific transcriptional activation of the glue genes; however, the receptor responsible for triggering this response has not been characterized. Here we show that the coordinated expression of the glue genes midway through the third instar is mediated by 20E acting to induce genes of the Broad Complex (BRC through a receptor that is not an EcR/USP heterodimer. This result is novel because it demonstrates for the first time that at least some 20E-mediated, mid-larval, developmental responses are controlled by an uncharacterized receptor that does not contain an RXR-like component.

  2. GRB2 Nucleates T Cell Receptor-Mediated LAT Clusters That Control PLC-γ1 Activation and Cytokine Production. (United States)

    Bilal, Mahmood Yousif; Houtman, Jon C D


    GRB2 is a ubiquitously expressed adaptor protein required for signaling downstream of multiple receptors. To address the role of GRB2 in receptor-mediated signaling, the expression of GRB2 was suppressed in human CD4+ T cells and its role downstream of the T cell receptor (TCR) was examined. Interestingly, GRB2 deficient T cells had enhanced signaling from complexes containing the TCR. However, GRB2 deficient T cells had substantially reduced production of IL-2 and IFN-γ. This defect was attributed to diminished formation of linker for activation of T cells (LAT) signaling clusters, which resulted in reduced MAP kinase activation, calcium flux, and PLC-γ1 recruitment to LAT signaling clusters. Add back of wild-type GRB2, but not a novel N-terminal SH3 domain mutant, rescued LAT microcluster formation, calcium mobilization, and cytokine release, providing the first direct evidence that GRB2, and its ability to bind to SH3 domain ligands, is required for establishing LAT microclusters. Our data demonstrate that the ability of GRB2 to facilitate protein clusters is equally important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes.

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


    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni


    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 loc...

  4. Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis (United States)

    Chandrashekaran, Varun; Das, Suvarthi; Seth, Ratanesh Kumar; Dattaroy, Diptadip; Alhasson, Firas; Michelotti, Gregory; Nagarkatti, Mitzi; Nagarkatti, Prakash; Diehl, Anna Mae; Chatterjee, Saurabh


    Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression. PMID:26474534

  5. Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway.

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    Laura W Bowers

    Full Text Available Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB: ≥30 kg/m2; normal weight (N: 18.5-24.9 kg/m2. Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231 and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease.

  6. Keratin 18 attenuates estrogen receptor α-mediated signaling by sequestering LRP16 in cytoplasm

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    Si Yiling


    Full Text Available Abstract Background Oncogenesis in breast cancer is often associated with excess estrogen receptor α(ERα activation and overexpression of its coactivators. LRP16 is both an ERα target gene and an ERα coactivator, and plays a crucial role in ERα activation and proliferation of MCF-7 breast cancer cells. However, the regulation of the functional availability of this coactivator protein is not yet clear. Results Yeast two-hybrid screening, GST pulldown and coimmunoprecipitation (CoIP identified the cytoplasmic intermediate filament protein keratin 18 (K18 as a novel LRP16-interacting protein. Fluorescence analysis revealed that GFP-tagged LRP16 was primarily localized in the nuclei of mock-transfected MCF-7 cells but was predominantly present in the cytoplasm of K18-transfected cells. Immunoblotting analysis demonstrated that the amount of cytoplasmic LRP16 was markedly increased in cells overexpressing K18 whereas nuclear levels were depressed. Conversely, knockdown of endogenous K18 expression in MCF-7 cells significantly decreased the cytoplasmic levels of LRP16 and increased levels in the nucleus. CoIP failed to detect any interaction between K18 and ERα, but ectopic expression of K18 in MCF-7 cells significantly blunted the association of LRP16 with ERα, attenuated ERα-activated reporter gene activity, and decreased estrogen-stimulated target gene expression by inhibiting ERα recruitment to DNA. Furthermore, BrdU incorporation assays revealed that K18 overexpression blunted the estrogen-stimulated increase of S-phase entry of MCF-7 cells. By contrast, knockdown of K18 in MCF-7 cells significantly increased ERα-mediated signaling and promoted cell cycle progression. Conclusions K18 can effectively associate with and sequester LRP16 in the cytoplasm, thus attenuating the final output of ERα-mediated signaling and estrogen-stimulated cell cycle progression of MCF-7 breast cancer cells. Loss of K18 increases the functional availability

  7. Roles of dopamine receptors in mediating acute modulation of immunological responses in Macrobrachium rosenbergii. (United States)

    Chang, Zhong-Wen; Ke, Zhi-Han; Chang, Chin-Chyuan


    Dopamine (DA) was found to influence the immunological responses and resistance to pathogen infection in invertebrates. To clarify the possible modulation of DA through dopamine receptors (DAR) against acute environmental stress, the levels of DA, glucose and lactate in the haemolymph of Macrobrachium rosenbergii under hypo- and hyperthermal stresses were measured. The changes in immune parameters such as total haemocyte count (THC), differential haemocyte count (DHC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities and phagocytic activity (PA) were evaluated in prawns which received DAR antagonists (SCH23390, SCH, D1 antagonist; domperidone, DOM, D2 antagonist; chlorpromazine, CH, D1+2 antagonist) followed by hypo- (15 °C) and hyperthermal (34 °C) stresses. In addition, pharmacological analysis of the effect DA modulation was studied in haemocytes incubated with DA and DAR antagonists. The results revealed a significant increase in haemolymph DA accompanied with upregulated levels of glucose and lactate in prawns exposed to both hypo- and hyperthermal stresses in 2 h. In addition, a significant decrease in RBs per haemocyte was noted in prawns which received DAR antagonists when they exposed to hyperthermal stress for 30 min. In in vitro test, antagonism on RBs, SOD and GPx activity of haemocytes were further evidenced through D1, D1, D1+D2 DARs, respectively, in the meantime, no significant difference in PO activity and PA was observed among the treatment groups. These results suggest that the upregulation of DA, glucose and lactate in haemolymph might be the response to acute thermal stress for the demand of energy, and the DAR occupied by its antagonistic action impart no effect on immunological responses except RBs in vivo even though the modulation mediated through D1 DAR was further evidenced in RBs, SOD and GPx activities in vitro. It is therefore concluded that thermal

  8. Purinergic receptor X7 mediates leptin induced GLUT4 function in stellate cells in nonalcoholic steatohepatitis. (United States)

    Chandrashekaran, Varun; Das, Suvarthi; Seth, Ratanesh Kumar; Dattaroy, Diptadip; Alhasson, Firas; Michelotti, Gregory; Nagarkatti, Mitzi; Nagarkatti, Prakash; Diehl, Anna Mae; Chatterjee, Saurabh


    Metabolic oxidative stress via CYP2E1 can act as a second hit in NASH progression. Our previous studies have shown that oxidative stress in NASH causes higher leptin levels and induces purinergic receptor X7 (P2X7r). We tested the hypothesis that higher circulating leptin due to CYP2E1-mediated oxidative stress induces P2X7r. P2X7r in turn activates stellate cells and causes increased proliferation via modulating Glut4, the glucose transporter, and increased intracellular glucose. Using a high fat diet-fed NAFLD model where bromodichloromethane (BDCM) was administered to induce CYP2E1-mediated oxidative stress, we show that P2X7r expression and protein levels were leptin and CYP2E1 dependent. P2X7r KO mice had significantly decreased stellate cell proliferation. Human NASH livers showed marked increase in P2X7r, and Glut4 in α-SMA positive cells. NASH livers had significant increase in Glut4 protein and phosphorylated AKT, needed for Glut4 translocation while leptin KO and P2X7r KO mice showed marked decrease in Glut4 levels primarily in stellate cells. Mechanistically stellate cells showed increase in phosphorylated AKT, Glut4 protein and localization in the membrane following administration of P2X7r agonist or leptin+P2X7r agonist, while use of P2X7r antagonist or AKT inhibitor attenuated the response suggesting that leptin-P2X7r axis in concert but not leptin alone is responsible for the Glut4 induction and translocation. Finally P2X7r-agonist and leptin caused an increase in intracellular glucose and consumption by increasing the activity of hexokinase. In conclusion, the study shows a novel role of leptin-induced P2X7r in modulating Glut4 induction and translocation in hepatic stellate cells, that are key to NASH progression.

  9. The specificity protein factor Sp1 mediates transcriptional regulation of P2X7 receptors in the nervous system. (United States)

    García-Huerta, Paula; Díaz-Hernandez, Miguel; Delicado, Esmerilda G; Pimentel-Santillana, María; Miras-Portugal, M Teresa; Gómez-Villafuertes, Rosa


    P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites.

  10. NMDA and PACAP receptor signaling interact to mediate retinal-induced scn cellular rhythmicity in the absence of light.

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    Ian C Webb

    Full Text Available The "core" region of the suprachiasmatic nucleus (SCN, a central clock responsible for coordinating circadian rhythms, shows a daily rhythm in phosphorylation of extracellular regulated kinase (pERK. This cellular rhythm persists under constant darkness and, despite the absence of light, is dependent upon inputs from the eye. The neural signals driving this rhythmicity remain unknown and here the roles of glutamate and PACAP are examined. First, rhythmic phosphorylation of the NR1 NMDA receptor subunit (pNR1, a marker for receptor activation was shown to coincide with SCN core pERK, with a peak at circadian time (CT 16. Enucleation and intraocular TTX administration attenuated the peak in the pERK and pNR1 rhythms, demonstrating that activation of the NMDA receptor and ERK in the SCN core at CT16 are dependent on retinal inputs. In contrast, ERK and NR1 phosphorylation in the SCN shell region were unaffected by these treatments. Intraventricular administration of the NMDA receptor antagonist MK-801 also attenuated the peak in SCN core pERK, indicating that ERK phosphorylation in this region requires NMDA receptor activation. As PACAP is implicated in photic entrainment and is known to modulate glutamate signaling, the effects of a PAC1 receptor antagonist (PACAP 6-38 on SCN core pERK and pNR1 also were examined. PACAP 6-38 administration attenuated SCN core pERK and pNR1, suggesting that PACAP induces pERK directly, and indirectly via a modulation of NMDA receptor signaling. Together, these data indicate that, in the absence of light, retinal-mediated NMDA and PAC1 receptor activation interact to induce cellular rhythms in the SCN core. These results highlight a novel function for glutamate and PACAP release in the hamster SCN apart from their well-known roles in the induction of photic circadian clock resetting.

  11. Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor.

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    Mostafa Zamanian

    Full Text Available G protein-coupled receptors (GPCRs represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o. The method was then extended to deorphanize a novel Gα(s-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

  12. Involvement of leukotriene B4 receptor 1 signaling in platelet-activating factor-mediated neutrophil degranulation and chemotaxis. (United States)

    Gaudreault, Eric; Stankova, Jana; Rola-Pleszczynski, Marek


    Platelet-activating factor (PAF) is a potent lipid mediator of inflammation that can act on human neutrophils. When neutrophils are stimulated with PAF at concentrations greater than 10 nM, a double peak of intracellular calcium mobilization is observed. The second calcium peak observed in PAF-treated neutrophils has already been suggested to come from the production of endogenous leukotriene B4 (LTB4). Here we demonstrate the involvement of endogenous LTB4 production and subsequent activation of the high affinity LTB4 receptor (BLT1) in this second calcium mobilization peak observed after stimulation with PAF. We also show that the second, but not the first peak, could be desensitized by prior exposure to LTB4. Moreover, when neutrophils were pre-treated with pharmacological inhibitors of LTB4 production or with the specific BLT1 antagonist, U75302, PAF-mediated neutrophil degranulation was inhibited by more than 50%. On the other hand, pre-treating neutrophils with the PAF receptor specific antagonist (WEB2086) did not prevent any LTB4-induced degranulation. Also, when human neutrophils were pre-treated with U75302, PAF-mediated chemotaxis was reduced by more than 60%. These data indicate the involvement of BLT1 signaling in PAF-mediated neutrophil activities.

  13. Histone H4 Lys 20 methyltransferase SET8 promotes androgen receptor-mediated transcription activation in prostate cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Lushuai [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yanyan; Du, Fengxia [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Han, Xiao [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Xiaohua [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China); Niu, Yuanjie [Chawnshang Chang Sex Hormone Research Center, Tianjin Institute of Urology, Tianjin Medical University, Tianjin 300070 (China); Ren, Shancheng, E-mail: [Department of Urology, Shanghai Changhai Hospital, Second Military Medical University, Shanghai 200433 (China); Sun, Yingli, E-mail: [Laboratory of Genome Variations and Precision Bio-Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101 (China)


    Highlights: • Dihydrotestosterone stimulates H4K20me1 enrichment at the PSA promoter. • SET8 promotes AR-mediated transcription activation. • SET8 interacts with AR and promotes cell proliferation. - Abstract: Histone methylation status in different lysine residues has an important role in transcription regulation. The effect of H4K20 monomethylation (H4K20me1) on androgen receptor (AR)-mediated gene transcription remains unclear. Here we show that AR agonist stimulates the enrichment of H4K20me1 and SET8 at the promoter of AR target gene PSA in an AR dependent manner. Furthermore, SET8 is crucial for the transcription activation of PSA. Co-immunoprecipitation analyses demonstrate that SET8 interacts with AR. Therefore, we conclude that SET8 is involved in AR-mediated transcription activation, possibly through its interaction with AR and H4K20me1 modification.

  14. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha. (United States)

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M; Hammock, Bruce; Tukey, Robert H


    Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ERα) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car(-/-) mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human

  15. Interaction of glucocorticoid receptor (GR) with estrogen receptor (ER) α and activator protein 1 (AP1) in dexamethasone-mediated interference of ERα activity. (United States)

    Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K


    The role of glucocorticoids in the inhibition of estrogen (17-β-estradiol (E2))-regulated estrogen receptor (ER)-positive breast cancer cell proliferation is well established. We and others have seen that synthetic glucocorticoid dexamethasone (Dex) antagonizes E2-stimulated endogenous ERα target gene expression. However, how glucocorticoids negatively regulate the ERα signaling pathway is still poorly understood. ChIP studies using ERα- and glucocorticoid receptor (GR)-positive MCF-7 cells revealed that GR occupies several ERα-binding regions (EBRs) in cells treated with E2 and Dex simultaneously. Interestingly, there was little or no GR loading to these regions when cells were treated with E2 or Dex alone. The E2+Dex-dependent GR recruitment is associated with the displacement of ERα and steroid receptor coactivator-3 from the target EBRs leading to the repression of ERα-mediated transcriptional activation. The recruitment of GR to EBRs requires assistance from ERα and FOXA1 and is facilitated by AP1 binding within the EBRs. The GR binding to EBRs is mediated via direct protein-protein interaction between the GR DNA-binding domain and ERα. Limited mutational analyses indicate that arginine 488 located within the C-terminal zinc finger domain of the GR DNA-binding domain plays a critical role in stabilizing this interaction. Together, the results of this study unravel a novel mechanism involved in glucocorticoid inhibition of ERα transcriptional activity and E2-mediated cell proliferation and thus establish a foundation for future exploitation of the GR signaling pathway in the treatment of ER-positive breast cancer.

  16. Activated niacin receptor HCA2 inhibits chemoattractant-mediated macrophage migration via Gβγ/PKC/ERK1/2 pathway and heterologous receptor desensitization (United States)

    Shi, Ying; Lai, Xiangru; Ye, Lingyan; Chen, Keqiang; Cao, Zheng; Gong, Wanghua; Jin, Lili; Wang, Chunyan; Liu, Mingyong; Liao, Yuan; Wang, Ji Ming; Zhou, Naiming


    The niacin receptor HCA2 is implicated in controlling inflammatory host responses with yet poorly understood mechanistic basis. We previously reported that HCA2 in A431 epithelial cells transduced Gβγ-protein kinase C- and Gβγ-metalloproteinase/EGFR-dependent MAPK/ERK signaling cascades. Here, we investigated the role of HCA2 in macrophage-mediated inflammation and the underlying mechanisms. We found that proinflammatory stimulants LPS, IL-6 and IL-1β up-regulated the expression of HCA2 on macrophages. Niacin significantly inhibited macrophage chemotaxis in response to chemoattractants fMLF and CCL2 by disrupting polarized distribution of F-actin and Gβ protein. Niacin showed a selected additive effect on chemoattractant-induced activation of ERK1/2, JNK and PI3K pathways, but only the MEK inhibitor UO126 reduced niacin-mediated inhibition of macrophage chemotaxis, while activation of ERK1/2 by EGF alone did not inhibit fMLF-mediated migration of HEK293T cells co-expressing HCA2 and fMLF receptor FPR1. In addition, niacin induced heterologous desensitization and internalization of FPR1. Furthermore, niacin rescued mice from septic shock by diminishing inflammatory symptoms and the effect was abrogated in HCA2−/− mice. These results suggest that Gβγ/PKC-dependent ERK1/2 activation and heterologous desensitization of chemoattractant receptors are involved in the inhibition of chemoattractant-induced migration of macrophages by niacin. Thus, HCA2 plays a critical role in host protection against pro-inflammatory insults. PMID:28186140

  17. Valerian inhibits rat hepatocarcinogenesis by activating GABA(A receptor-mediated signaling.

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    Anna Kakehashi

    Full Text Available Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA A receptor (GABA(AR system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(AR agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN. Formation of glutathione S-transferase placental form positive (GST-P(+ foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2'-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P(+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21(Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(AR alpha 1 subunit was observed in GST-P(+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P(+ foci by activating GABA(AR-mediated signaling.

  18. Forkhead box A3 mediates glucocorticoid receptor function in adipose tissue. (United States)

    Ma, Xinran; Xu, Lingyan; Mueller, Elisabetta


    Glucocorticoids (GCs) are widely prescribed anti-inflammatory agents, but their chronic use leads to undesirable side effects such as excessive expansion of adipose tissue. We have recently shown that the forkhead box protein A3 (Foxa3) is a calorie-hoarding factor that regulates the selective enlargement of epididymal fat depots and suppresses energy expenditure in a nutritional- and age-dependent manner. It has been demonstrated that Foxa3 levels are elevated in adipose depots in response to high-fat diet regimens and during the aging process; however no studies to date have elucidated the mechanisms that control Foxa3's expression in fat. Given the established effects of GCs in increasing visceral adiposity and in reducing thermogenesis, we assessed the existence of a possible link between GCs and Foxa3. Computational prediction analysis combined with molecular studies revealed that Foxa3 is regulated by the glucocorticoid receptor (GR) in preadipocytes, adipocytes, and adipose tissues and is required to facilitate the binding of the GR to its target gene promoters in fat depots. Analysis of the long-term effects of dexamethasone treatment in mice revealed that Foxa3 ablation protects mice specifically against fat accretion but not against other pathological side effects elicited by this synthetic GC in tissues such as liver, muscle, and spleen. In conclusion our studies provide the first demonstration, to our knowledge, that Foxa3 is a direct target of GC action in adipose tissues and point to a role of Foxa3 as a mediator of the side effects induced in fat tissues by chronic treatment with synthetic steroids.

  19. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  20. Insulin receptors mediate growth effects in cultured fetal neurons. I. Rapid stimulation of protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Heidenreich, K.A.; Toledo, S.P. (Univ. of California-San Diego, La Jolla (USA))


    In this study we have examined the effects of insulin on protein synthesis in cultured fetal chick neurons. Protein synthesis was monitored by measuring the incorporation of (3H)leucine (3H-leu) into trichloroacetic acid (TCA)-precipitable protein. Upon addition of 3H-leu, there was a 5-min lag before radioactivity occurred in protein. During this period cell-associated radioactivity reached equilibrium and was totally recovered in the TCA-soluble fraction. After 5 min, the incorporation of 3H-leu into protein was linear for 2 h and was inhibited (98%) by the inclusion of 10 micrograms/ml cycloheximide. After 24 h of serum deprivation, insulin increased 3H-leu incorporation into protein by approximately 2-fold. The stimulation of protein synthesis by insulin was dose dependent (ED50 = 70 pM) and seen within 30 min. Proinsulin was approximately 10-fold less potent than insulin on a molar basis in stimulating neuronal protein synthesis. Insulin had no effect on the TCA-soluble fraction of 3H-leu at any time and did not influence the uptake of (3H)aminoisobutyric acid into neurons. The isotope ratio of 3H-leu/14C-leu in the leucyl tRNA pool was the same in control and insulin-treated neurons. Analysis of newly synthesized proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that insulin uniformly increased the incorporation of 14C-leu into all of the resolved neuronal proteins. We conclude from these data that (1) insulin rapidly stimulates overall protein synthesis in fetal neurons independent of amino acid uptake and aminoacyl tRNA precursor pools; (2) stimulation of protein synthesis is mediated by the brain subtype of insulin receptor; and (3) insulin is potentially an important in vivo growth factor for fetal central nervous system neurons.

  1. Androgen receptor serine 81 phosphorylation mediates chromatin binding and transcriptional activation. (United States)

    Chen, Shaoyong; Gulla, Sarah; Cai, Changmeng; Balk, Steven P


    Our previous findings indicated that androgen receptor (AR) phosphorylation at serine 81 is stimulated by the mitotic cyclin-dependent kinase 1 (CDK1). In this report, we extended our previous study and confirmed that Ser-81 phosphorylation increases during mitosis, coincident with CDK1 activation. We further showed blocking cell cycle at G(1) or S phase did not disrupt androgen-induced Ser-81 phosphorylation and AR-dependent transcription, consistent with a recent report that AR was phosphorylated at Ser-81 and activated by the transcriptional CDK9. To assess the function of Ser-81 phosphorylation in prostate cancer (PCa) cells expressing endogenous AR, we developed a ligand switch strategy using a ligand-binding domain mutation (W741C) that renders AR responsive to the antagonist bicalutamide. An S81A/W741C double mutant AR stably expressed in PCa cells failed to transactivate the endogenous AR-regulated PSA or TMPRSS2 genes. ChIP showed that the S81A mutation prevented ligand-induced AR recruitment to these genes, and cellular fractionation revealed that the S81A mutation globally abrogated chromatin binding. Conversely, the AR fraction rapidly recruited to chromatin after androgen stimulation was highly enriched for Ser-81 phosphorylation. Finally, inhibition of CDK1 and CDK9 decreased AR Ser-81 phosphorylation, chromatin binding, and transcriptional activity. These findings indicate that Ser-81 phosphorylation by CDK9 stabilizes AR chromatin binding for transcription and suggest that CDK1-mediated Ser-81 phosphorylation during mitosis provides a pool of Ser-81 phosphorylation AR that can be readily recruited to chromatin for gene reactivation and may enhance AR activity in PCa.

  2. Aging-like skin changes in metabolic syndrome model mice are mediated by mineralocorticoid receptor signaling. (United States)

    Nagase, Takashi; Akase, Tomoko; Sanada, Hiromi; Minematsu, Takeo; Ibuki, Ai; Huang, Lijuan; Asada, Mayumi; Yoshimura, Kotaro; Nagase, Miki; Shimada, Tsutomu; Aburada, Masaki; Nakagami, Gojiro; Sugama, Junko


    Aging is accelerated, at least in part, by pathological condition such as metabolic syndrome (MetS), and various molecular pathways such as oxidative stress are common mediators of aging and MetS. We previously developed the aging-like skin model by single ultraviolet (UV) irradiation on the MetS model mice. Recent studies revealed that mineralocorticoid receptor (MR) signaling plays a pivotal role for various tissue inflammation and damages in MetS. Although previous studies reported that MR is expressed in the skin and that overexpression of MR in the skin resulted in the skin atrophy, the physiological or pathological functions of MR in the skin are not fully elucidated. Here, we show the involvement of MR signaling in the aging-like skin changes in our own model. Elevations of oxidative stress and inflammation markers were observed in the MetS mice, and the UV-evoked aging-like skin damages were attenuated by topical antioxidant. MR expression was higher in the MetS mouse skin, and notably, expression of its effecter gene Sgk1 was significantly upregulated in the aging-like skin in the UV-irradiated MetS mice. Furthermore, topical application of MR antagonist spironolactone suppressed Sgk1 expression, oxidative stress, inflammation, and the aging-like changes in the skin. The 2-week UV onto the non-MetS mice, the more usual photoaging model, resulted in the skin damages mostly equivalent to the MetS mice with single UV, but they were not associated with upregulation of MR signaling. Our studies suggested an unexpected role of MR signaling in the skin aging in MetS status.

  3. Fcgamma receptor-mediated suppression of human immunodeficiency virus type 1 replication in primary human macrophages. (United States)

    Perez-Bercoff, Danielle; David, Annie; Sudry, Hugues; Barré-Sinoussi, Françoise; Pancino, Gianfranco


    Permissiveness of monocytes and macrophages to human immunodeficiency virus (HIV) infection is modulated by various stimuli. In this study we demonstrate that stimulation of primary monocytes and monocyte-derived macrophages (MDM) through the receptors for the Fc portion of immunoglobulin G (IgG) (FcgammaR) inhibits HIV type 1 (HIV-1) replication. Viral p24 production was decreased by 1.5 to 3 log units in MDM infected with both R5 and X4 HIV-1 strains upon stimulation by immobilized IgG but not upon stimulation by soluble IgG or by F(ab')(2) IgG fragments. Although MDM activation by immobilized IgG induced high levels of macrophage-derived chemokine secretion as well as a sustained down-regulation of CD4 and a transient decrease in CCR5 expression, these factors did not appear to play a major role in the suppression of HIV-1 replication. Single-cycle infection of FcgammaR-stimulated MDM with HIV-1 virions pseudotyped with either HIV-1 R5 or vesicular stomatitis virus G envelopes was inhibited, suggesting a postentry restriction of viral replication. PCR analyses of HIV-1 DNA intermediate replication forms suggested that reverse transcription is not affected by stimulation with immobilized human IgG, at least during the first replication cycle. The accumulation of PCR products corresponding to nuclear unintegrated two-long-terminal-repeat circles and the relative decrease of integrated HIV-1 DNA signals suggest an inhibition of proviral integration. Our data, showing that FcgammaR-mediated activation of MDM is a potent mechanism of HIV-1 suppression, raise the possibility that FcgammaR cross-linking by immune complexes may contribute to the control of viral replication in macrophages.

  4. LYN-activating mutations mediate antiestrogen resistance in estrogen receptor-positive breast cancer. (United States)

    Schwarz, Luis J; Fox, Emily M; Balko, Justin M; Garrett, Joan T; Kuba, María Gabriela; Estrada, Mónica Valeria; González-Angulo, Ana María; Mills, Gordon B; Red-Brewer, Monica; Mayer, Ingrid A; Abramson, Vandana; Rizzo, Monica; Kelley, Mark C; Meszoely, Ingrid M; Arteaga, Carlos L


    Estrogen receptor-positive (ER(+)) breast cancers adapt to hormone deprivation and become resistant to antiestrogen therapy. Here, we performed deep sequencing on ER(+) tumors that remained highly proliferative after treatment with the aromatase inhibitor letrozole and identified a D189Y mutation in the inhibitory SH2 domain of the SRC family kinase (SFK) LYN. Evaluation of 463 breast tumors in The Cancer Genome Atlas revealed four LYN mutations, two of which affected the SH2 domain. In addition, LYN was upregulated in multiple ER(+) breast cancer lines resistant to long-term estrogen deprivation (LTED). An RNAi-based kinome screen revealed that LYN is required for growth of ER(+) LTED breast cancer cells. Kinase assays and immunoblot analyses of SRC substrates in transfected cells indicated that LYN(D189Y) has higher catalytic activity than WT protein. Further, LYN(D189Y) exhibited reduced phosphorylation at the inhibitory Y507 site compared with LYN(WT). Other SH2 domain LYN mutants, E159K and K209N, also exhibited higher catalytic activity and reduced inhibitory site phosphorylation. LYN(D189Y) overexpression abrogated growth inhibition by fulvestrant and/or the PI3K inhibitor BKM120 in 3 ER(+) breast cancer cell lines. The SFK inhibitor dasatinib enhanced the antitumor effect of BKM120 and fulvestrant against estrogen-deprived ER(+) xenografts but not LYN(D189Y)-expressing xenografts. These results suggest that LYN mutations mediate escape from antiestrogens in a subset of ER(+) breast cancers.

  5. Sphingosine-1-phosphate receptor 3 mediates sphingosine-1-phosphate induced release of weibel-palade bodies from endothelial cells.

    Directory of Open Access Journals (Sweden)

    Kathinka W E M van Hooren

    Full Text Available Sphingosine-1-phosphate (S1P is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs. S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1 mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1. S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.

  6. Sphingosine-1-phosphate receptor 3 mediates sphingosine-1-phosphate induced release of weibel-palade bodies from endothelial cells. (United States)

    van Hooren, Kathinka W E M; Spijkers, Léon J A; van Breevoort, Dorothee; Fernandez-Borja, Mar; Bierings, Ruben; van Buul, Jaap D; Alewijnse, Astrid E; Peters, Stephan L M; Voorberg, Jan


    Sphingosine-1-phosphate (S1P) is an agonist for five distinct G-protein coupled receptors, that is released by platelets, mast cells, erythrocytes and endothelial cells. S1P promotes endothelial cell barrier function and induces release of endothelial cell-specific storage-organelles designated Weibel-Palade bodies (WPBs). S1P-mediated enhancement of endothelial cell barrier function is dependent on S1P receptor 1 (S1PR1) mediated signaling events that result in the activation of the small GTPase Rac1. Recently, we have reported that Rac1 regulates epinephrine-induced WPB exocytosis following its activation by phosphatidylinositol-3,4,5-triphosphate-dependent Rac exchange factor 1 (PREX1). S1P has also been described to induce WPB exocytosis. Here, we confirm that S1P induces release of WPBs using von Willebrand factor (VWF) as a marker. Using siRNA mediated knockdown of gene expression we show that S1PR1 is not involved in S1P-mediated release of WPBs. In contrast depletion of the S1PR3 greatly reduced S1P-induced release of VWF. S1P-mediated enhancement of endothelial barrier function was not affected by S1PR3-depletion whereas it was greatly impaired in cells lacking S1PR1. The Rho kinase inhibitor Y27632 completely abrogated S1P-mediated release of VWF. Also, the calcium chelator BAPTA-AM significantly reduced S1P-induced release of VWF. Our findings indicate that S1P-induced release of haemostatic, inflammatory and angiogenic components stored within WPBs depends on the S1PR3.

  7. Estrogen receptor-related receptor alpha mediates up-regulation of aromatase expression by prostaglandin E2 in prostate stromal cells. (United States)

    Miao, Lin; Shi, Jiandang; Wang, Chun-Yu; Zhu, Yan; Du, Xiaoling; Jiao, Hongli; Mo, Zengnan; Klocker, Helmut; Lee, Chung; Zhang, Ju


    Estrogen receptor-related receptor alpha (ERRalpha) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRalpha is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRalpha, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRalpha, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRalpha expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRalpha activity by chlordane (an antagonist of ERRalpha) or small interfering RNA knockdown of ERRalpha blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRalpha significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRalpha directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRalpha and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17Beta-estradiol concentration in WPMY-1 medium was up-regulated by ERRalpha expression, and that was further increased by PGE2. Our results provided evidence that ERRalpha contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRalpha in estrogen-related prostatic diseases.

  8. Receptor-mediated gene delivery using polyethylenimine (PEI)coupled with polypeptides targeting FGF receptors on cells surface

    Institute of Scientific and Technical Information of China (English)

    LI Da; WANG Qing-qing; TANG Gu-ping; HUANG Hong-liang; SHEN Fen-ping; LI Jing-zhong; YU Hai


    Objective: To construct a novel kind ofnonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods:The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides including DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. Conclusion: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer gene therapy.

  9. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression. (United States)

    Scarlatti, G; Tresoldi, E; Björndal, A; Fredriksson, R; Colognesi, C; Deng, H K; Malnati, M S; Plebani, A; Siccardi, A G; Littman, D R; Fenyö, E M; Lusso, P


    Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain

  10. Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-labouring myometrium.

    LENUS (Irish Health Repository)

    Fanning, Rebecca A


    The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1 × 10(-7)M-1 × 10(-4)M; 54% reduction in contractile activity, P<0.001 at 1 × 10(-4)M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro.

  11. Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-labouring myometrium. (United States)

    Fanning, Rebecca A; McMorrow, Jason P; Campion, Deirdre P; Carey, Michael F; O'Connor, John J


    The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1 × 10(-7)M-1 × 10(-4)M; 54% reduction in contractile activity, Popioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro.

  12. Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations (United States)

    Li, Zhenlong; Gorfe, Alemayehu A.


    Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

  13. The Src homology 3 binding domain is required for lysophosphatidic acid 3 receptor-mediated cellular viability in melanoma cells. (United States)

    Jia, Wei; Tran, Sterling K; Ruddick, Caitlin A; Murph, Mandi M


    The LPA3 receptor is a G protein-coupled receptor that binds extracellular lysophosphatidic acid and mediates intracellular signaling cascades. Although we previously reported that receptor inhibition using siRNA or chemical inhibition obliterates the viability of melanoma cells, the mechanism was unclear. Herein we hypothesized that amino acids comprising the Src homology 3 (SH3) ligand binding motif, R/K-X-X-V/P-X-X-P or (216)-KTNVLSP-(222), within the third intracellular loop of LPA3 were critical in mediating this outcome. Therefore, we performed site-directed mutagenesis of the lysine, valine and proline, replacing these amino acids with alanines, and evaluated the changes in viability, proliferation, ERK1/2 signaling and calcium in response to lysophosphatidic acid. Our results show that enforced LPA3 expression in SK-MEL-2 cells enhanced their resiliency by allowing these cells to oppose any loss of viability during growth in serum-free medium for up to 96 h, in contrast to parental SK-MEL-2 cells, which show a significant decline in viability. Similarly, site-directed alanine substitutions of valine and proline, V219A/P222A or 2aa-SK-MEL-2 cells, did not significantly alter viability, but adding a further alanine to replace the lysine, K216A/V219A/P222A or 3aa-SK-MEL-2 cells, obliterated this function. In addition, an inhibitor of the LPA3 receptor had no impact on the parental SK-MEL-2, 2aa-SK-MEL-2 or 3aa-SK-MEL-2 cells, but significantly reduced viability among wt-LPA3-SK-MEL-2 cells. Taken together, the data suggest that the SH3 ligand binding domain of LPA3 is required to mediate viability in melanoma cells.

  14. The differential effects of 5-HT(1A) receptor stimulation on dopamine receptor-mediated abnormal involuntary movements and rotations in the primed hemiparkinsonian rat. (United States)

    Dupre, Kristin B; Eskow, Karen L; Negron, Giselle; Bishop, Christopher


    Serotonin 1A receptor (5-HT(1A)R) agonists have emerged as valuable supplements to l-DOPA therapy, demonstrating that they can decrease side effects and enhance motor function in animal models of Parkinson's disease (PD) and human PD patients. The precise mechanism by which these receptors act remains unknown and there is limited information on how 5-HT(1A)R stimulation impacts striatal dopamine (DA) D1 receptor (D1R) and D2 receptor (D2R) function. The current study examined the effects of 5-HT(1A)R stimulation on DA receptor-mediated behaviors. Male Sprague-Dawley rats were rendered hemiparkinsonian by unilateral 6-OHDA lesions and primed with the D1R agonist SKF81297 (0.8 mg/kg, i.p.) in order to sensitize DA receptors. Using a randomized within subjects design, rats received a first injection of: Vehicle (dH(2)O) or the 5-HT(1A)R agonist +/-8-OH-DPAT (0.1 or 1.0 mg/kg, i.p.), followed by a second injection of: Vehicle (dimethyl sulfoxide), the D1R agonist SKF81297 (0.8 mg/kg, i.p.), the D2R agonist quinpirole (0.2 mg/kg, i.p.), or l-DOPA (12 mg/kg+benserazide, 15 mg/kg, i.p.). On test days, rats were monitored over a 2-h period immediately following the second injection for abnormal involuntary movements (AIMs), analogous to dyskinesia observed in PD patients, and contralateral rotations. The present findings indicate that 5-HT(1A)R stimulation reduces AIMs induced by D1R, D2R and l-DOPA administration while its effects on DA agonist-induced rotations were receptor-dependent, suggesting that direct 5-HT(1A)R and DA receptor interactions may contribute to the unique profile of 5-HT(1A)R agonists for the improvement of PD treatment.

  15. TRIM32 promotes retinoic acid receptor {alpha}-mediated differentiation in human promyelogenous leukemic cell line HL60

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Tomonobu [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Okumura, Fumihiko [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan); Iguchi, Akihiro; Ariga, Tadashi [Department of Pediatrics, Hokkaido University Graduate School of Medicine, Sapporo 060-8638 (Japan); Hatakeyama, Shigetsugu, E-mail: [Department of Biochemistry, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido 060-8638 (Japan)


    Highlights: Black-Right-Pointing-Pointer TRIM32 enhanced RAR{alpha}-mediated transcriptional activity even in the absence of RA. Black-Right-Pointing-Pointer TRIM32 stabilized RAR{alpha} in the human promyelogenous leukemic cell line HL60. Black-Right-Pointing-Pointer Overexpression of TRIM32 in HL60 cells induced granulocytic differentiation. Black-Right-Pointing-Pointer TRIM32 may function as a coactivator for RAR{alpha}-mediated transcription in APL cells. -- Abstract: Ubiquitination, one of the posttranslational modifications, appears to be involved in the transcriptional activity of nuclear receptors including retinoic acid receptor {alpha} (RAR{alpha}). We previously reported that an E3 ubiquitin ligase, TRIM32, interacts with several important proteins including RAR{alpha} and enhances transcriptional activity of RAR{alpha} in mouse neuroblastoma cells and embryonal carcinoma cells. Retinoic acid (RA), which acts as a ligand to nuclear receptors including RAR{alpha}, plays crucial roles in development, differentiation, cell cycles and apoptosis. In this study, we found that TRIM32 enhances RAR{alpha}-mediated transcriptional activity even in the absence of RA and stabilizes RAR{alpha} in the human promyelogenous leukemic cell line HL60. Moreover, we found that overexpression of TRIM32 in HL60 cells suppresses cellular proliferation and induces granulocytic differentiation even in the absence of RA. These findings suggest that TRIM32 functions as one of the coactivators for RAR{alpha}-mediated transcription in acute promyelogenous leukemia (APL) cells, and thus TRIM32 may become a potentially therapeutic target for APL.

  16. Anaesthetic impairment of immune function is mediated via GABA(A receptors.

    Directory of Open Access Journals (Sweden)

    Daniel W Wheeler

    Full Text Available GABA(A receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.We demonstrate, using RT-PCR, that monocytes express GABA(A receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.Our results show that functional GABA(A receptors are present on monocytes with properties similar to CNS GABA(A receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life

  17. Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons. (United States)

    Torres-Torrelo, Julio; Torres, Blas; Carrascal, Livia


    The neuronal input-output function depends on recruitment threshold and gain of the firing frequency-current (f-I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input-output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f-I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f-I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

  18. Characterization of GABA/sub A/ receptor-mediated /sup 36/chloride uptake in rat brain synaptoneurosomes

    Energy Technology Data Exchange (ETDEWEB)

    Luu, M.D.; Morrow, A.L.; Paul, S.M.; Schwartz, R.D.


    ..gamma..-Aminobutyric acid (GABA) receptor-mediated /sup 36/chloride (/sup 36/Cl/sup -/) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated /sup 36/Cl/sup -/ uptake in a concentration-dependent manner with the following order of potency: Muscimol>GABA>piperidine-4-sulfonic acid (P4S)>4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP)=3-aminopropanesulfonic acid (3APS)>>taurine. Both P4S and 3APS behaved as partial agonists, while the GABA/sub B/ agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated /sup 36/Cl/sup -/ uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated /sup 36/Cl/sup -/ uptake was also dependent on the anion present in the media. The muscinol response varied in media containing the following anions: Br/sup -/>Cl/sup -/greater than or equal toNO/sub 3//sup -/>I/sup -/greater than or equal toSCN/sup -/>>C/sub 3/H/sub 5/OO/sup -/greater than or equal toClO/sub 4//sup -/>F/sup -/, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl/sup -/ channel. 43 references, 4 figures, 3 tables.

  19. Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein. (United States)

    FitzGerald, D J; Fryling, C M; Zdanovsky, A; Saelinger, C B; Kounnas, M; Winkles, J A; Strickland, D; Leppla, S


    The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 14-2-1 line had significant amounts of LRP, but in contrast to wild-type cells, little or no receptor was displayed on the cell surface. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin

  20. The role of NMDA and non-NMDA receptors in the NTS in mediating three distinct sympathoinhibitory reflexes. (United States)

    Sartor, Daniela M; Verberne, Anthony J M


    Cholecystokinin (CCK) elicits a sympathetic vasomotor reflex that is implicated in gastrointestinal circulatory control. We sought to determine (1) the site in the solitary tract nucleus (NTS) responsible for mediating this reflex and (2) the possible involvement of excitatory amino acid (EAA) receptors. In addition, we sought to determine whether the NTS site responsible for mediating the baroreflex (phenylephrine, PE, 10 microg/kg i.v.) and the von Bezold-Jarisch reflex (phenylbiguanide, PBG, 10 microg/kg i.v) overlap with that involved in the CCK-induced reflex (CCK, 4 microg/kg, i.v.), and to compare the relative importance of NMDA and non-NDMA receptors in these reflexes. In separate experiments, the effects of PE, PBG, and CCK on mean arterial blood pressure, heart rate, and splanchnic sympathetic nerve discharge were tested before and after bilateral microinjection into the NTS of the gamma-aminobutyric acid(A) (GABA(A)) agonist muscimol, the EAA antagonist kynurenate, the NMDA receptor antagonist D: (-)-2-amino-5-phosphopentanoic acid (AP-5), the non-NMDA receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), AP-5 + NBQX, or vehicle. While all treatments (except vehicle) significantly attenuated/abolished/reversed the splanchnic sympathoinhibitory responses to PE, PBG, and CCK, the extent of blockade varied between the different treatment groups. Both NMDA and non-NMDA receptors were essential to the baroreflex and the von Bezold-Jarisch reflex, whereas the CCK reflex was more dependent on non-NMDA receptors. Muscimol, kynurenate, and AP-5 + NBQX significantly attenuated the bradycardic responses to PE and PBG (P NTS responsible for eliciting all three reflexes, NMDA and non-NMDA receptors are recruited differentially for the full expression of these reflexes. The CCK-induced sympathoinhibitory reflex is unique in that it relies predominantly on non-NMDA receptors in the NTS and elicits bradycardic effects that

  1. DMPD: Signal transduction pathways mediated by the interaction of CpG DNA withToll-like receptor 9. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 14751759 Signal transduction pathways mediated by the interaction of CpG DNA withTo...;16(1):17-22. (.png) (.svg) (.html) (.csml) Show Signal transduction pathways mediated by the interaction of... CpG DNA withToll-like receptor 9. PubmedID 14751759 Title Signal transduction pathways media

  2. The muscarinic acetylcholine receptor agonist BuTAC mediates antipsychotic-like effects via the M4 subtype. (United States)

    Watt, Marla L; Rorick-Kehn, Linda; Shaw, David B; Knitowski, Karen M; Quets, Anne T; Chesterfield, Amy K; McKinzie, David L; Felder, Christian C


    The generation of muscarinic acetylcholine receptor (mAChR) subtype-selective compounds has been challenging, requiring use of nonpharmacological approaches, such as genetically engineered animals, to deepen our understanding of the potential that members of the muscarinic receptor subtype family hold as therapeutic drug targets. The muscarinic receptor agonist 'BuTAC' was previously shown to exhibit efficacy in animal models of psychosis, although the particular receptor subtype(s) responsible for such activity was unclear. Here, we evaluate the in vitro functional agonist and antagonist activity of BuTAC using an assay that provides a direct measure of G protein activation. In addition, we employ the conditioned avoidance response paradigm, an in vivo model predictive of antipsychotic activity, and mouse genetic deletion models to investigate which presynaptic mAChR subtype mediates the antipsychotic-like effects of BuTAC. Our results show that, in vitro, BuTAC acts as a full agonist at the M2AChR and a partial agonist at the M1 and M4 receptors, with full antagonist activity at M3- and M5AChRs. In the mouse conditioned avoidance response (CAR) assay, BuTAC exhibits an atypical antipsychotic-like profile by selectively decreasing avoidance responses at doses that do not induce escape failures. CAR results using M2(-/-), M4(-/-), and M2/M4 (M2/M4(-/-)) mice found that the effects of BuTAC were near completely lost in M2/M4(-/-) double-knockout mice and potency of BuTAC was right-shifted in M4(-/-) as compared with wild-type and M2(-/-) mice. The M2/M4(-/-) mice showed no altered sensitivity to the antipsychotic effects of either haloperidol or clozapine, suggesting that these compounds mediate their actions in CAR via a non-mAChR-mediated mechanism. These data support a role for the M4AChR subtype in mediating the antipsychotic-like activity of BuTAC and implicate M4AChR agonism as a potential novel therapeutic mechanism for ameliorating symptoms associated with

  3. Phenobarbital but not diazepam reduces AMPA/Kainate receptor mediated currents and exerts opposite actions on initial seizures in the neonatal rat hippocampus

    Directory of Open Access Journals (Sweden)

    Romain eNardou


    Full Text Available 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 3 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 Depolarising Potentials suggesting that PB may exert additional actions that are not mediated by GABA signalling. 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 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 of neonatal seizures.

  4. Pregnane X Receptor (PXR)-Mediated Gene Repression and Cross-Talk of PXR with Other Nuclear Receptors via Coactivator Interactions (United States)

    Pavek, Petr


    Pregnane X receptor is a ligand-activated nuclear receptor (NR) that mainly controls inducible expression of xenobiotics handling genes including biotransformation enzymes and drug transporters. Nowadays it is clear that PXR is also involved in regulation of intermediate metabolism through trans-activation and trans-repression of genes controlling glucose, lipid, cholesterol, bile acid, and bilirubin homeostasis. In these processes PXR cross-talks with other NRs. Accumulating evidence suggests that the cross-talk is often mediated by competing for common coactivators or by disruption of coactivation and activity of other transcription factors by the ligand-activated PXR. In this respect mainly PXR-CAR and PXR-HNF4α interference have been reported and several cytochrome P450 enzymes (such as CYP7A1 and CYP8B1), phase II enzymes (SULT1E1, Gsta2, Ugt1a1), drug and endobiotic transporters (OCT1, Mrp2, Mrp3, Oatp1a, and Oatp4) as well as intermediate metabolism enzymes (PEPCK1 and G6Pase) have been shown as down-regulated genes after PXR activation. In this review, I summarize our current knowledge of PXR-mediated repression and coactivation interference in PXR-controlled gene expression regulation. PMID:27932985

  5. CRM 1-mediated degradation and agonist-induced down-regulation of beta-adrenergic receptor mRNAs. (United States)

    Bai, Ying; Lu, Huafei; Machida, Curtis A


    The beta1-adrenergic receptor (beta1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3' untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308-1324], we examined the agonist-mediated down-regulation of the rat beta1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing beta1-ARs in the brain, results in significant decreases in beta1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in beta1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the beta1-AR 3' UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of beta1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the beta1-AR mRNAs.

  6. Fc receptors do not mediate African swine fever virus replication in macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Alcami, A.; Vinuela, E. (Centro de Biologia Molecular, Universidad Autonoma, Madrid (Spain))


    Titration experiments in swine macrophages have shown that African swine fever virus infectivity was not enhanced in the presence of antiviral antibodies. The early viral protein synthesis and the viral DNA replication in swine macrophages infected with virus-antibody complexes were inhibited in the presence of high doses of uv-inactivated virus, which saturated specific virus receptors, but not when Fc receptors were saturated with antibodies. These results indicate that African swine fever virus does not infect swine macrophages through Fc receptors and that the normal entry pathway through virus receptors is not bypassed by the virus-antibody complexes.

  7. Positive inotropy mediated via CGRP receptors in isolated human myocardial trabeculae

    DEFF Research Database (Denmark)

    Saetrum Opgaard, O; Hasbak, P; de Vries, R;


    Isometric contractile force were studied on isolated human myocardial trabeculae that were paced at 1.0 Hz in tissue baths. Alpha calcitonin gene-related peptide (alpha-CGRP) had a potent positive inotropic effect in most trabeculae from both the right atrium and left ventricle, and this effect...... reaction (PCR) mRNAs encoding the human calcitonin receptor-like receptor and the receptor associated modifying proteins (RAMPs) RAMP1, RAMP2, and RAMP3 were detected in human myocardial trabeculae from both the right atrium and left ventricle. In conclusion, functional CGRP(1) and CGRP(2) receptors may...

  8. The 5-HT7 receptor as a mediator and modulator of antidepressant-like behavior


    Sarkisyan, Gor; Roberts, Amanda J.; Hedlund, Peter B.


    The 5-HT7 receptor has been suggested as a target for treating depression since inactivation or blockade of the receptor has an antidepressant-like behavioral effect. The present study investigated possible interactions between various classes of drugs with antidepressant properties and blockade or inactivation of the 5-HT7 receptor. Immobility despair in the tail suspension test and the forced swim test was evaluated in mice lacking the 5-HT7 receptor (5-HT7−/−) and in wild-type controls (5-...

  9. Molecular Mechanisms Mediating Nociceptin/Orphanin FQ Receptor Signaling, Desensitization and Internalization. (United States)

    Baiula, Monica; Bedini, Andrea; Carbonari, Gioia


    In 1994, the isolation of an opioid receptor-related clone soon led to the isolation and characterization of a novel neuropeptide, termed nociceptin or orphanin FQ (N/OFQ). This heptadecapeptide binds to the N/OFQ receptor (NOP) with high affinity, but does not interact directly with classical opioid receptors. The regional distribution of N/OFQ and of its receptor suggest any possible involvement of this neurotransmission system in motor and balance control, reinforcement and reward, nociception, stress response, sexual behavior, aggression and autonomic control of physiological processes as well as of immune functions. The actions of N/OFQ may also be uniquely dependent on contextual factors, both genetic and environmental. As for most of the G protein coupled receptors, NOP C-terminal sequences are believed to interact with proteins that are mandatory for anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Increasing details of how NOP receptors are activated and removed from the plasma membrane have been elucidated in vitro, and more importantly in a physiological context. Details of how these receptors travel and recycle following internalization have also shed light on the importance of such mechanisms for any potential therapeutic use of NOP ligands. A picture of the pathways and proteins involved in these processes is beginning to emerge. This review will address molecular events contributing to NOP receptor signaling and trafficking.

  10. Mu-Opioid (MOP) receptor mediated G-protein signaling is impaired in specific brain regions in a rat model of schizophrenia. (United States)

    Szűcs, Edina; Büki, Alexandra; Kékesi, Gabriella; Horváth, Gyöngyi; Benyhe, Sándor


    Schizophrenia is a complex mental health disorder. Clinical reports suggest that many patients with schizophrenia are less sensitive to pain than other individuals. Animal models do not interpret schizophrenia completely, but they can model a number of symptoms of the disease, including decreased pain sensitivities and increased pain thresholds of various modalities. Opioid receptors and endogenous opioid peptides have a substantial role in analgesia. In this biochemical study we investigated changes in the signaling properties of the mu-opioid (MOP) receptor in different brain regions, which are involved in the pain transmission, i.e., thalamus, olfactory bulb, prefrontal cortex and hippocampus. Our goal was to compare the transmembrane signaling mediated by MOP receptors in control rats and in a recently developed rat model of schizophrenia. Regulatory G-protein activation via MOP receptors were measured in [(35)S]GTPγS binding assays in the presence of a highly selective MOP receptor peptide agonist, DAMGO. It was found that the MOP receptor mediated activation of G-proteins was substantially lower in membranes prepared from the 'schizophrenic' model rats than in control animals. The potency of DAMGO to activate MOP receptor was also decreased in all brain regions studied. Taken together in our rat model of schizophrenia, MOP receptor mediated G-proteins have a reduced stimulatory activity compared to membrane preparations taken from control animals. The observed distinct changes of opioid receptor functions in different areas of the brain do not explain the augmented nociceptive threshold described in these animals.

  11. Antitumor activities and on-target toxicities mediated by a TRAIL receptor agonist following cotreatment with panobinostat. (United States)

    Martin, Ben P; Frew, Ailsa J; Bots, Michael; Fox, Stephen; Long, Fenella; Takeda, Kazuyoshi; Yagita, Hideo; Atadja, Peter; Smyth, Mark J; Johnstone, Ricky W


    The recent development of novel targeted anticancer therapeutics such as histone deacetylase inhibitors (HDACi) and activators of the TRAIL pathway provide opportunities for the introduction of new treatment regimens in oncology. HDACi and recombinant TRAIL or agonistic anti-TRAIL receptor antibodies have been shown to induce synergistic tumor cell apoptosis and some therapeutic activity in vivo. Herein, we have used syngeneic preclinical models of human solid cancers to demonstrate that the HDACi panobinostat can sensitize tumor cells to apoptosis mediated by the anti-mouse TRAIL receptor antibody MD5-1. We demonstrate that the combination of panobinostat and MD5-1 can eradicate tumors grown subcutaneously and orthotopically in immunocompetent mice, while single agent treatment has minimal effect. However, escalation of the dose of panobinostat to enhance antitumor activity resulted in on-target MD5-1-mediated gastrointestinal toxicities that were fatal to the treated mice. Studies performed in mice with knockout of the TRAIL receptor showed that these mice could tolerate doses of the panobinostat/MD5-1 combination that were lethal in wild type mice resulting in superior tumor clearance. Given that clinical studies using HDACi and activators of the TRAIL pathway have been initiated, our preclinical data highlight the potential toxicities that could limit the use of such a treatment regimen. Our studies also demonstrate the power of using syngeneic in vivo tumor models as physiologically relevant preclinical systems to test the antitumor effects and identify potential side effects of novel anticancer regimens.

  12. The CB1 receptor mediates the peripheral effects of ghrelin on AMPK activity but not on growth hormone release. (United States)

    Kola, Blerina; Wittman, Gábor; Bodnár, Ibolya; Amin, Faisal; Lim, Chung Thong; Oláh, Márk; Christ-Crain, Mirjam; Lolli, Francesca; van Thuijl, Hinke; Leontiou, Chrysanthia A; Füzesi, Tamás; Dalino, Paolo; Isidori, Andrea M; Harvey-White, Judith; Kunos, George; Nagy, György M; Grossman, Ashley B; Fekete, Csaba; Korbonits, Márta


    This study aimed to investigate whether the growth hormone release and metabolic effects of ghrelin on AMPK activity of peripheral tissues are mediated by cannabinoid receptor type 1 (CB1) and the central nervous system. CB1-knockout (KO) and/or wild-type mice were injected peripherally or intracerebroventricularly with ghrelin and CB1 antagonist rimonabant to study tissue AMPK activity and gene expression (transcription factors SREBP1c, transmembrane protein FAS, enzyme PEPCK, and protein HSL). Growth hormone levels were studied both in vivo and in vitro. Peripherally administered ghrelin in liver, heart, and adipose tissue AMPK activity cannot be observed in CB1-KO or CB1 antagonist-treated mice. Intracerebroventricular ghrelin treatment can influence peripheral AMPK activity. This effect is abolished in CB1-KO mice and by intracerebroventricular rimonabant treatment, suggesting that central CB1 receptors also participate in the signaling pathway that mediates the effects of ghrelin on peripheral tissues. Interestingly, in vivo or in vitro growth hormone release is intact in response to ghrelin in CB1-KO animals. Our data suggest that the metabolic effects of ghrelin on AMPK in peripheral tissues are abolished by the lack of functional CB1 receptor via direct peripheral effect and partially through the central nervous system, thus supporting the existence of a possible ghrelin-cannabinoid-CB1-AMPK pathway.

  13. Metabotropic P2Y1 receptor signalling mediates astrocytic hyperactivity in vivo in an Alzheimer's disease mouse model. (United States)

    Delekate, Andrea; Füchtemeier, Martina; Schumacher, Toni; Ulbrich, Cordula; Foddis, Marco; Petzold, Gabor C


    Astrocytic network alterations have been reported in Alzheimer's disease (AD), but the underlying pathways have remained undefined. Here we measure astrocytic calcium, cerebral blood flow and amyloid-β plaques in vivo in a mouse model of AD using multiphoton microscopy. We find that astrocytic hyperactivity, consisting of single-cell transients and calcium waves, is most pronounced in reactive astrogliosis around plaques and is sometimes associated with local blood flow changes. We show that astroglial hyperactivity is reduced after P2 purinoreceptor blockade or nucleotide release through connexin hemichannels, but is augmented by increasing cortical ADP concentration. P2X receptor blockade has no effect, but inhibition of P2Y1 receptors, which are strongly expressed by reactive astrocytes surrounding plaques, completely normalizes astrocytic hyperactivity. Our data suggest that astroglial network dysfunction is mediated by purinergic signalling in reactive astrocytes, and that intervention aimed at P2Y1 receptors or hemichannel-mediated nucleotide release may help ameliorate network dysfunction in AD.

  14. Pharmacological evidence for the mediation of the panicolytic effect of fluoxetine by dorsal periaqueductal gray matter μ-opioid receptors. (United States)

    Roncon, Camila Marroni; Almada, Rafael Carvalho; Maraschin, Jhonatan Christian; Audi, Elisabeth Aparecida; Zangrossi, Hélio; Graeff, Frederico Guilherme; Coimbra, Norberto Cysne


    Previously reported results have shown that the inhibitory effect of fluoxetine on escape behavior, interpreted as a panicolytic-like effect, is blocked by pretreatment with either the opioid receptor antagonist naloxone or the 5-HT1A receptor (5-HT1A-R) antagonist WAY100635 via injection into the dorsal periaqueductal gray matter (dPAG). Additionally, reported evidence indicates that the μ-opioid receptor (MOR) interacts with the 5-HT1A-R in the dPAG. In the present work, pretreatment of the dPAG with the selective MOR blocker CTOP antagonized the anti-escape effect of chronic fluoxetine (10 mg/kg, i.p., daily, for 21 days), as measured in the elevated T-maze (ETM) test, indicating mediation of this effect by the MOR. In addition, the combined administration of sub-effective doses of the selective MOR agonist DAMGO (intra-dPAG) and sub-effective doses of chronic as well as subchronic (7 days) fluoxetine increased avoidance and escape latencies, suggesting that the activation of MORs may facilitate and accelerate the effects of fluoxetine. The current observation that MORs located in the dPAG mediate the anti-escape effect of fluoxetine may open new perspectives for the development of more efficient and fast-acting panic-alleviating drugs.

  15. Adenylyl cyclase-associated protein 1 is a receptor for human resistin and mediates inflammatory actions of human monocytes. (United States)

    Lee, Sahmin; Lee, Hyun-Chae; Kwon, Yoo-Wook; Lee, Sang Eun; Cho, Youngjin; Kim, Joonoh; Lee, Soobeom; Kim, Ju-Young; Lee, Jaewon; Yang, Han-Mo; Mook-Jung, Inhee; Nam, Ky-Youb; Chung, Junho; Lazar, Mitchell A; Kim, Hyo-Soo


    Human resistin is a cytokine that induces low-grade inflammation by stimulating monocytes. Resistin-mediated chronic inflammation can lead to obesity, atherosclerosis, and other cardiometabolic diseases. Nevertheless, the receptor for human resistin has not been clarified. Here, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We found that human resistin directly binds to CAP1 in monocytes and upregulates cyclic AMP (cAMP) concentration, protein kinase A (PKA) activity, and NF-κB-related transcription of inflammatory cytokines. Overexpression of CAP1 in monocytes enhanced the resistin-induced increased activity of the cAMP-dependent signaling. Moreover, CAP1-overexpressed monocytes aggravated adipose tissue inflammation in transgenic mice that express human resistin from their monocytes. In contrast, suppression of CAP1 expression abrogated the resistin-mediated inflammatory activity both in vitro and in vivo. Therefore, CAP1 is the bona fide receptor for resistin leading to inflammation in humans.

  16. Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples

    Energy Technology Data Exchange (ETDEWEB)

    Hilscherova, K. [Dept. of Environmental Chemistry and Toxicology, Faculty of Science, Masaryk Univ., Brno (Czech Republic); Machala, M. [Veterinary Research Inst. of Veterinary Medicine, Brno (Czech Republic); Kannan, K.; Giesy, J.P. [Dept. of Zoology, National Food Safety and Toxicology Center, Inst. for Environmental Toxicology, Michigan State Univ., East Lansing, MI (United States); Blankenship, A.L. [Dept. of Zoology, National Food Safety and Toxicology Center, Inst. for Environmental Toxicology, Michigan State Univ., East Lansing, MI (United States); ENTRIX Inc., East Lansing, MI (United States)


    In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (AhR)- (hereafter referred as dioxin-like) or estrogen receptor (ER)-mediated activities of potential endocrine disrupting compounds (EDCs) in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQs) or estrogenic equivalents (E{sub 2}-EQs) and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Mass-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation. (orig.)

  17. 5-HT7 receptor-mediated fear conditioning and possible involvement of extracellular signal-regulated kinase. (United States)

    Takeda, Kotaro; Tsuji, Minoru; Miyagawa, Kazuya; Takeda, Hiroshi


    Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The present study examined the involvement of extracellular signal-regulated kinase 1/2 (ERK) signaling on the serotonin (5-HT)7 receptor-mediated fear conditioning. Conditioning was performed in a trial in which a tone was followed by an electrical foot-shock. Context- and tone-dependent fear were examined in tests conducted 24 and 48h after conditioning, respectively. The selective 5-HT7 receptor antagonist 2a-[4-(4-phenyl-1,2,3,6-tetrahydropyridyl)butyl]-2a,3,4,-tetrahydrobenzo(c,d)indol-2-(1H)-one (DR4004) (5mg/kg), when administered intraperitoneally (i.p.) immediately after conditioning, caused a significant decrease in both context- and tone-dependent fear responses (freezing behavior). A significant increase in ERK activity was observed in the amygdala of mice that displayed context- or tone-dependent fear responses, and these changes were also inhibited by the administration of DR4004 (5mg/kg, i.p.) immediately after conditioning. In contrast, the increase in hippocampal ERK activity in mice that displayed context-dependent fear responses was further enhanced by the administration of DR4004 (5mg/kg, i.p.). These results suggest that 5-HT7 receptor-mediated ERK signaling may play a significant role in the processes of emotional learning and memory.

  18. The Pyrexia transient receptor potential channel mediates circadian clock synchronization to low temperature cycles in Drosophila melanogaster. (United States)

    Wolfgang, Werner; Simoni, Alekos; Gentile, Carla; Stanewsky, Ralf


    Circadian clocks are endogenous approximately 24 h oscillators that temporally regulate many physiological and behavioural processes. In order to be beneficial for the organism, these clocks must be synchronized with the environmental cycles on a daily basis. Both light : dark and the concomitant daily temperature cycles (TCs) function as Zeitgeber ('time giver') and efficiently entrain circadian clocks. The temperature receptors mediating this synchronization have not been identified. Transient receptor potential (TRP) channels function as thermo-receptors in animals, and here we show that the Pyrexia (Pyx) TRP channel mediates temperature synchronization in Drosophila melanogaster. Pyx is expressed in peripheral sensory organs (chordotonal organs), which previously have been implicated in temperature synchronization. Flies deficient for Pyx function fail to synchronize their behaviour to TCs in the lower range (16-20°C), and this deficit can be partially rescued by introducing a wild-type copy of the pyx gene. Synchronization to higher TCs is not affected, demonstrating a specific role for Pyx at lower temperatures. In addition, pyx mutants speed up their clock after being exposed to TCs. Our results identify the first TRP channel involved in temperature synchronization of circadian clocks.

  19. Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells. (United States)

    Hoffmann, Kristina; Xifró, Rosa Altarcheh; Hartweg, Julia Lisa; Spitzlei, Petra; Meis, Kirsten; Molderings, Gerhard J; von Kügelgen, Ivar


    The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 μM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

  20. Mediation of autophagic cell death by type 3 ryanodine receptor (RyR3 in adult hippocampal neural stem cells

    Directory of Open Access Journals (Sweden)

    Kyung Min eChung


    Full Text Available Cytoplasmic Ca2+ actively engages in diverse intracellular processes from protein synthesis, folding and trafficking to cell survival and death. Dysregulation of intracellular Ca2+ levels is observed in various neuropathological states including Alzheimer’s and Parkinson’s diseases. Ryanodine receptors (RyRs and IP3 receptors (IP3Rs, the main Ca2+ release channels located in endoplasmic reticulum (ER membranes, are known to direct various cellular events such as autophagy and apoptosis. Here we investigated the intracellular Ca2+-mediated regulation of survival and death of adult hippocampal neural stem (HCN cells utilizing an insulin withdrawal model of autophagic cell death. Despite comparable expression levels of RyR and IP3R transcripts in HCN cells at normal state, the expression levels of RyRs — especially RyR3 — were markedly upregulated upon insulin withdrawal. While treatment with the RyR agonist caffeine significantly promoted the autophagic death of insulin-deficient HCN cells, treatment with its inhibitor dantrolene prevented the induction of autophagy following insulin withdrawal. Furthermore, CRISPR/Cas9-mediated knockout of the RyR3 gene abolished autophagic cell death of HCN cells. This study delineates a distinct, RyR3-mediated ER Ca2+ regulation of autophagy and programmed cell death in neural stem cells. Our findings provide novel insights into the critical, yet understudied mechanisms underlying the regulatory function of ER Ca2+ in neural stem cell biology.

  1. Orphan G protein-coupled receptor GPRC5A modulates integrin β1-mediated epithelial cell adhesion. (United States)

    Bulanova, Daria R; Akimov, Yevhen A; Rokka, Anne; Laajala, Teemu D; Aittokallio, Tero; Kouvonen, Petri; Pellinen, Teijo; Kuznetsov, Sergey G


    G-Protein Coupled Receptor (GPCR), Class C, Group 5, Member A (GPRC5A) has been implicated in several malignancies. The underlying mechanisms, however, remain poorly understood. Using a panel of human cell lines, we demonstrate that CRISPR/Cas9-mediated knockout and RNAi-mediated depletion of GPRC5A impairs cell adhesion to integrin substrates: collagens I and IV, fibronectin, as well as to extracellular matrix proteins derived from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Matrigel). Consistent with the phenotype, knock-out of GPRC5A correlated with a reduced integrin β1 (ITGB1) protein expression, impaired phosphorylation of the focal adhesion kinase (FAK), and lower activity of small GTPases RhoA and Rac1. Furthermore, we provide the first evidence for a direct interaction between GPRC5A and a receptor tyrosine kinase EphA2, an upstream regulator of FAK, although its contribution to the observed adhesion phenotype is unclear. Our findings reveal an unprecedented role for GPRC5A in regulation of the ITGB1-mediated cell adhesion and it's downstream signaling, thus indicating a potential novel role for GPRC5A in human epithelial cancers.

  2. Identification and molecular characterization of cellular factors required for glucocorticoid receptor-mediated mRNA decay (United States)

    Park, Ok Hyun; Park, Joori; Yu, Mira; An, Hyoung-Tae; Ko, Jesang; Kim, Yoon Ki


    Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.

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

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    Ma, Zhiyuan, E-mail: [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Yu, Yijun, E-mail: [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Tang, Song [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Liu, Hongling, E-mail: [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Su, Guanyong; Xie, Yuwei [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Giesy, John P. [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Hecker, Markus [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Yu, Hongxia [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China)


    Highlights: • Effects of TBOEP on expression of genes of several nuclear hormone receptors and their relationship with adverse effect pathways in zebrafish. • TBOEP was neither an agonist nor antagonist of AR or AhR as determined by use of in vitro mammalian cell-based receptor transactivation assays. • Modulation of ER- and MR-dependent pathways allowed for development of feasible receptor-mediated, critical mechanisms of toxic action. - Abstract: 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 0.5 μM 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 120 hpf. Exposure to 0.5 μM 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

  4. Repeated morphine treatment-mediated hyperalgesia, allodynia and spinal glial activation are blocked by co-administration of a selective cannabinoid receptor type-2 agonist


    Tumati, Suneeta; Largent-Milnes, Tally M.; Keresztes, Attila; Ren, Jiyang; Roeske, William R.; Vanderah, Todd W; Varga, Eva V.


    Spinal glial activation has been implicated in sustained morphine-mediated paradoxical pain sensitization. Since activation of glial CB2 cannabinoid receptors attenuates spinal glial activation in neuropathies, we hypothesized that CB2 agonists may also attenuate sustained morphine–mediated spinal glial activation and pain sensitization. Our data indicate that co-administration of a CB2-selective agonist (AM 1241) attenuates morphine (intraperitoneal; twice daily; 6 days)-mediated thermal hyp...

  5. Neural cell adhesion molecule-180-mediated homophilic binding induces epidermal growth factor receptor (EGFR) down-regulation and uncouples the inhibitory function of EGFR in neurite outgrowth

    DEFF Research Database (Denmark)

    Povlsen, Gro Klitgaard; Berezin, Vladimir; Bock, Elisabeth


    The neural cell adhesion molecule (NCAM) plays important roles in neuronal development, regeneration, and synaptic plasticity. NCAM homophilic binding mediates cell adhesion and induces intracellular signals, in which the fibroblast growth factor receptor plays a prominent role. Recent studies...... not require NCAM-mediated fibroblast growth factor receptor activation....... on axon guidance in Drosophila suggest that NCAM also regulates the epidermal growth factor receptor (EGFR) (Molecular and Cellular Neuroscience, 28, 2005, 141). A possible interaction between NCAM and EGFR in mammalian cells has not been investigated. The present study demonstrates for the first time...

  6. Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

    Directory of Open Access Journals (Sweden)

    Hildebrand Dagmar


    Full Text Available Abstract Background Lipopolysaccharide (LPS-triggered Toll-like receptor (TLR 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune

  7. Leptin-induced increase in leukemia inhibitory factor and its receptor by human endometrium is partially mediated by interleukin 1 receptor signaling. (United States)

    Gonzalez, R R; Rueda, B R; Ramos, M P; Littell, R D; Glasser, S; Leavis, P C


    Leptin and leukemia inhibitory factor (LIF) have been implicated as important mediators of implantation. The present study was designed to investigate whether leptin can directly regulate the expression of LIF and its receptor (LIF-R) in human endometrial cells and/or whether leptin-induced effects are linked to, or regulated in part by IL-1 signaling. Primary endometrial cells and endometrial epithelial cell lines (HES and Ishikawa cells) were cultured for 24-48 h in a medium containing insulin (5 microg/ml) and leptin (3, 10, and 62 nm) or IL-1beta (0.6, 3, and 10 nm) in the presence or absence of cytokines and/or receptor antagonists. The endpoints included phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the relative levels of LIF, LIF-R, IL-1beta, IL-1 receptor antagonist (IL-1Ra) and IL-1 receptor type I (IL-1R tI) as determined by ELISA or Western blotting techniques. Leptin treatment increases the level of phosphorylated STAT3, LIF-R, and LIF. Leptin also increases the levels of IL-1 ligand, receptor, and antagonist as was previously reported. Blockade of OB-R with antibodies or with a specific OB-R inhibitor (leptin peptide antagonist-2) abrogated leptin-induced effects, suggesting that leptin binding to its receptor activates Janus kinase 2/STAT3 signaling. Treatment of endometrial cells with IL-1beta also results in elevated levels of LIF-R. Interestingly, the inhibition of IL-1R tI with a specific antibody or with IL-1Ra negatively affects both leptin-induced and IL-1-induced effects on LIF-R levels. Abnormal endometrial LIF expression has been associated with human infertility and leptin has profound effects on the levels of LIF, IL-1, and their cognate receptors in vitro. Thus, it is tempting to speculate that leptin's role in vivo could include the regulation of other key cytokines to be fundamental to endometrial receptivity during implantation (i.e. LIF and IL-1).

  8. Nuclear exportin receptor CAS regulates the NPI-1-mediated nuclear import of HIV-1 Vpr.

    Directory of Open Access Journals (Sweden)

    Eri Takeda

    Full Text Available Vpr, an accessory protein of human immunodeficiency virus type 1, is a multifunctional protein that plays an important role in viral replication. We have previously shown that the region between residues 17 and 74 of Vpr (Vpr(N17C74 contained a bona fide nuclear localization signal and it is targeted Vpr(N17C74 to the nuclear envelope and then imported into the nucleus by importin α (Impα alone. The interaction between Impα and Vpr is important not only for the nuclear import of Vpr but also for HIV-1 replication in macrophages; however, it was unclear whether full-length Vpr enters the nucleus in a manner similar to Vpr(N17C74. This study investigated the nuclear import of full-length Vpr using the three typical Impα isoforms, Rch1, Qip1 and NPI-1, and revealed that full-length Vpr is selectively imported by NPI-1, but not Rch1 and Qip1, after it makes contact with the perinuclear region in digitonin-permeabilized cells. A binding assay using the three Impα isoforms showed that Vpr bound preferentially to the ninth armadillo repeat (ARM region (which is also essential for the binding of CAS, the export receptor for Impα in all three isoforms. Comparison of biochemical binding affinities between Vpr and the Impα isoforms using surface plasmon resonance analysis demonstrated almost identical values for the binding of Vpr to the full-length isoforms and to their C-terminal domains. By contrast, the data showed that, in the presence of CAS, Vpr was released from the Vpr/NPI-1 complex but was not released from Rch1 or Qip1. Finally, the NPI-1-mediated nuclear import of Vpr was greatly reduced in semi-intact CAS knocked-down cells and was recovered by the addition of exogenous CAS. This report is the first to show the requirement for and the regulation of CAS in the functioning of the Vpr-Impα complex.

  9. Downstream Toll-like receptor signaling mediates adaptor-specific cytokine expression following focal cerebral ischemia

    Directory of Open Access Journals (Sweden)

    Bolanle Famakin


    Full Text Available Abstract Background Deletion of some Toll-like receptors (TLRs affords protection against cerebral ischemia, but disruption of their known major downstream adaptors does not. To determine whether compensation in the production of downstream effectors by one pathway when the other is disrupted can explain these findings, we examined cytokine/chemokine expression and inflammatory infiltrates in wild-type (WT, MyD88−/− and TRIF-mutant mice following permanent middle cerebral artery occlusion (pMCAO. Methods Cytokine/chemokine expression was measured with a 25-plex bead array in the serum and brains of all three groups of mice at baseline (no surgery/naïve and at 3 hours and 24 hours following pMCAO. Brain inflammatory and neutrophil infiltrates were examined 24 hours following pMCAO. Results IL-6, keratinocyte chemoattractant (KC, granulocyte colony-stimulating factor (G-CSF and IL-10 were significantly decreased in MyD88−/− mice compared to WT mice following pMCAO. Significantly, decreased levels of the neutrophil chemoattractants KC and G-CSF corresponded with a trend toward fewer neutrophils in the brains of MyD88−/− mice. IP-10 was significantly decreased when either pathway was disrupted. MIP-1α was significantly decreased in TRIF-mutant mice, consistent with TRIF-dependent production. MyD88−/− mice showed elevations of a number of Th2 cytokines, such as IL-13, at baseline, which became significantly decreased following pMCAO. Conclusions Both MyD88 and TRIF mediate pathway-specific cytokine production following focal cerebral ischemia. Our results also suggest a compensatory Th2-type skew at baseline in MyD88−/− mice and a paradoxical switch to a Th1 phenotype following focal cerebral ischemia. The MyD88 pathway directs the expression of neutrophil chemoattractants following cerebral ischemia.

  10. Membrane coordination of receptors and channels mediating the inhibition of neuronal ion currents by ADP. (United States)

    Gafar, Hend; Dominguez Rodriguez, Manuel; Chandaka, Giri K; Salzer, Isabella; Boehm, Stefan; Schicker, Klaus


    ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.

  11. Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

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    Silva, Jose Alberto Alves da


    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  12. Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

    DEFF Research Database (Denmark)

    Møller, Cathrine Laustrup; Raun, Kirsten; Jacobsen, Marianne Lambert


    The melanocortin receptors (MCRs) belong to the G-protein coupled receptors (family A). So far, 5 different subtypes have been described (MC1R-MC5R) and of these MC2R and MC5R have been proposed to act directly in adipocytes and regulate lipolysis in rodents. Using ACTH and a-melanocyte stimulating...

  13. A3 Adenosine receptors mediate oligodendrocyte death and ischemic damage to optic nerve. (United States)

    González-Fernández, Estíbaliz; Sánchez-Gómez, María Victoria; Pérez-Samartín, Alberto; Arellano, Rogelio O; Matute, Carlos


    Adenosine receptor activation is involved in myelination and in apoptotic pathways linked to neurodegenerative diseases. In this study, we investigated the effects of adenosine receptor activation in the viability of oligodendrocytes of the rat optic nerve. Selective activation of A3 receptors in pure cultures of oligodendrocytes caused concentration-dependent apoptotic and necrotic death which was preceded by oxidative stress and mitochondrial membrane depolarization. Oligodendrocyte apoptosis induced by A3 receptor activation was caspase-dependent and caspase-independent. In addition to dissociated cultures, incubation of optic nerves ex vivo with adenosine and the A3 receptor agonist 2-CI-IB-MECA(1-[2-Chloro-6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide)-induced caspase-3 activation, oligodendrocyte damage, and myelin loss, effects which were prevented by the presence of caffeine and the A3 receptor antagonist MRS 1220 (N-[9-Chloro-2-(2-furanyl)[1,2,4]-triazolo [1,5-c]quinazolin-5-yl]benzene acetamide). Finally, ischemia-induced injury and functional loss to the optic nerve was attenuated by blocking A3 receptors. Together, these results indicate that adenosine may trigger oligodendrocyte death via activation of A3 receptors and suggest that this mechanism contributes to optic nerve and white matter ischemic damage.

  14. P2X receptors mediate ATP-induced primary nociceptive neurone activation. (United States)

    Bland-Ward, P A; Humphrey, P P


    ATP-gated P2X ion-channel receptors are localised throughout the mammalian nervous system and have been identified on neurones which participate in conduction of nociceptive information from the periphery to, and within, the CNS. This article briefly reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X(3) receptor in this process. The P2X(3) receptor subunit is almost exclusively expressed on a subset of small and medium diameter sensory neurones innervating cutaneous and visceral tissue. Activation of P2X receptors present on the peripheral terminals of primary afferents results in neuronal depolarisation and, in conscious animals, leads to the manifestation of acute nociceptive behaviour. Recent animal studies have also shown that P2X(3) receptor expression is increased in sensory ganglia following acute neuronal injury, hinting that similar plasticity in the expression of this receptor subtype could underlie the mechanisms involved in a range of conditions characterised by sensory hypersensitivity in man. It is apparent from the evidence available that functional antagonists at specific P2X receptor subtypes could represent an important class of novel analgesic agents.

  15. Complement-mediated solubilization of immune complexes and their interaction with complement C3 receptors

    DEFF Research Database (Denmark)

    Petersen, Ivan; Baatrup, Gunnar; Jepsen, H H;


    Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components of the me......Some of the molecular events in the complement (C)-mediated solubilization of immune complexes (IC) have been clarified in recent years. The solubilization is primarily mediated by alternative C pathway proteins whereas factors in the classical pathway accelerate the process. Components...

  16. Cannabinoid receptor 2-mediated attenuation of CXCR4-tropic HIV infection in primary CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Cristina Maria Costantino

    Full Text Available Agents that activate cannabinoid receptor pathways have been tested as treatments for cachexia, nausea or neuropathic pain in HIV-1/AIDS patients. The cannabinoid receptors (CB(1R and CB(2R and the HIV-1 co-receptors, CCR5 and CXCR4, all signal via Gαi-coupled pathways. We hypothesized that drugs targeting cannabinoid receptors modulate chemokine co-receptor function and regulate HIV-1 infectivity. We found that agonism of CB(2R, but not CB(1R, reduced infection in primary CD4+ T cells following cell-free and cell-to-cell transmission of CXCR4-tropic virus. As this change in viral permissiveness was most pronounced in unstimulated T cells, we investigated the effect of CB(2R agonism on to CXCR4-induced signaling following binding of chemokine or virus to the co-receptor. We found that CB(2R agonism decreased CXCR4-activation mediated G-protein activity and MAPK phosphorylation. Furthermore, CB(2R agonism altered the cytoskeletal architecture of resting CD4+ T cells by decreasing F-actin levels. Our findings suggest that CB(2R activation in CD4+ T cells can inhibit actin reorganization and impair productive infection following cell-free or cell-associated viral acquisition of CXCR4-tropic HIV-1 in resting cells. Therefore, the clinical use of CB(2R agonists in the treatment of AIDS symptoms may also exert beneficial adjunctive antiviral effects against CXCR4-tropic viruses in late stages of HIV-1 infection.

  17. Cannabinoid receptor CB1 mediates baseline and activity-induced survival of new neurons in adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Müller Anke


    Full Text Available Abstract Background Adult neurogenesis is a particular example of brain plasticity that is partially modulated by the endocannabinoid system. Whereas the impact of synthetic cannabinoids on the neuronal progenitor cells has been described, there has been lack of information about the action of plant-derived extracts on neurogenesis. Therefore we here focused on the effects of Δ9-tetrahydrocannabinol (THC and Cannabidiol (CBD fed to female C57Bl/6 and Nestin-GFP-reporter mice on proliferation and maturation of neuronal progenitor cells and spatial learning performance. In addition we used cannabinoid receptor 1 (CB1 deficient mice and treatment with CB1 antagonist AM251 in Nestin-GFP-reporter mice to investigate the role of the CB1 receptor in adult neurogenesis in detail. Results THC and CBD differed in their effects on spatial learning and adult neurogenesis. CBD did not impair learning but increased adult neurogenesis, whereas THC reduced learning without affecting adult neurogenesis. We found the neurogenic effect of CBD to be dependent on the CB1 receptor, which is expressed over the whole dentate gyrus. Similarly, the neurogenic effect of environmental enrichment and voluntary wheel running depends on the presence of the CB1 receptor. We found that in the absence of CB1 receptors, cell proliferation was increased and neuronal differentiation reduced, which could be related to CB1 receptor mediated signaling in Doublecortin (DCX-expressing intermediate progenitor cells. Conclusion CB1 affected the stages of adult neurogenesis that involve intermediate highly proliferative progenitor cells and the survival and maturation of new neurons. The pro-neurogenic effects of CBD might explain some of the positive therapeutic features of CBD-based compounds.

  18. NK3 receptors mediate an increase in firing rate of midbrain dopamine neurons of the rat and the guinea pig. (United States)

    Werkman, Taco R; McCreary, Andrew C; Kruse, Chris G; Wadman, Wytse J


    This in vitro study investigates and compares the effects of NK3 receptor ligands on the firing rate of rat and guinea pig midbrain dopamine neurons. The findings are discussed in the light of choosing suitable animal models for investigating pharmacological properties of NK3 receptor antagonists, which have been proposed to possess therapeutic activity in neuropsychiatric diseases like e.g. schizophrenia. In vitro midbrain slice preparations of both species were used to record (extracellularly) the firing rates of dopamine neurons located in the substantia nigra (SN) and ventral tegmental area (VTA). Furthermore, the effect of the D2 receptor agonist quinpirole on guinea pig SN and VTA dopamine neurons was investigated. The efficacy of quinpirole in inhibiting guinea pig dopamine neuron firing activity was much less as compared to that of rat dopamine neurons, suggesting a lower dopamine D2 autoreceptor density on the guinea pig neurons. The NK3 receptor agonist senktide induced in subpopulations of rat SN (55%) and VTA (79%) and guinea pig SN (50%) and VTA (21%) dopamine neurons an increase in firing rate. In responsive neurons this effect was concentration-dependent with EC₅₀ values of 3-5 nM (for both species). The selective NK3 receptor antagonist osanetant (100 nM) was able to partly block the senktide-induced increase in firing rates of dopamine neurons and shifted the concentration-response relation curves for senktide to the right (pA₂ values were ~7.5). The fractional block of the senktide responses by osanetant appeared to be larger in guinea pig dopamine neurons, indicating that osanetant is a more potent blocker of NK3 receptor-mediated responses with noncompetitive properties in the guinea pig.

  19. Toll-like receptor 8 ligands activate a vitamin D mediated autophagic response that inhibits human immunodeficiency virus type 1. (United States)

    Campbell, Grant R; Spector, Stephen A


    Toll-like receptors (TLR) are important in recognizing microbial pathogens and triggering host innate immune responses, including autophagy, and in the mediation of immune activation during human immunodeficiency virus type-1 (HIV) infection. We report here that TLR8 activation in human macrophages induces the expression of the human cathelicidin microbial peptide (CAMP), the vitamin D receptor (VDR) and cytochrome P450, family 27, subfamily B, polypeptide 1 (CYP27B1), which 1α-hydroxylates the inactive form of vitamin D, 25-hydroxycholecalciferol, into its biologically active metabolite. Moreover, we demonstrate using RNA interference, chemical inhibitors and vitamin D deficient media that TLR8 agonists inhibit HIV through a vitamin D and CAMP dependent autophagic mechanism. These data support an important role for vitamin D in the control of HIV infection, and provide a biological explanation for the benefits of vitamin D. These findings also provide new insights into potential novel targets to prevent and treat HIV infection.

  20. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)


    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  1. Timosaponin AIII induces antiplatelet and antithrombotic activity via Gq-mediated signaling by the thromboxane A2 receptor (United States)

    Cong, Yue; Wang, Limei; Peng, Renjun; Zhao, Yang; Bai, Fan; Yang, Chao; Liu, Xiaolan; Wang, Daqian; Ma, Baiping; Cong, Yuwen


    The thromboxane (Tx) A2 pathway is a major contributor to the amplification of initial platelet activation and is therefore a key drug target. To identify potent small-molecule inhibitors of the thromboxane prostaglandin (TP) receptor, we screened a small steroidal saponin library using U46619-induced rat platelet aggregation assays. Timosaponin AIII (TAIII) was identified as a potent inhibitor of U46619-induced rat platelet aggregation and exhibited superior selectivity for the TP receptor versus other G protein-coupled receptors and a PKC activator. TAIII inhibited U46619-induced rat platelet aggregation independent of increases in cAMP and cGMP and the inhibition of TxA2 production. Both PKC and PLC activators restored TAIII-inhibited platelet aggregation, whereas TAIII did not inhibit platelet aggregation induced by co-activation of the G12/13 and Gz pathways. Furthermore, TAIII did not affect the platelet shape change or ROCK2 phosphorylation evoked by low-dose U46619. In vivo, TAIII prolonged tail bleeding time, reduced the mortality of animals with acute pulmonary thromboembolism and significantly reduced venous thrombus weight. Our study suggests that TAIII, by preferentially targeting Gq-mediated PLC/PKC signaling from the TP receptor, induces stronger in vitro antiplatelet activity and in vivo antithrombotic effects and may be an excellent candidate for the treatment of thrombotic disorders. PMID:27934923

  2. Detection of Interaction of Binding Affinity of Aromatic Hydrocarbon Receptor to the Specific DNA by Exonuclease Protection Mediated PCR Assay

    Institute of Scientific and Technical Information of China (English)

    SUN Xi; XU Shunqing


    A novel exonuclease protection mediated PCR assay (EPM-PCR) to detect the interaction of protein and DNA at a dioxin-responsive enhancer (DRE) upstream of the CYP1A1 gene in rat hepatic cytosol was established. A double-stranded DNA fragment containing two binding sites was designed and incubated with the aryl hydrocarbon receptor (AhR) transformed by 2,3,7,8-tet rachlorodibenzo p dioxin (TCDD) to generate TCDD: AhR: DNA complex which could protect receptor-binding DNA against exonuclease Ⅲ (Exo Ⅲ) digestion. With ExoⅢ treatment, free DNAs were digested and receptor-bound DNAs remained that could be amplified by PCR. By agarose gel electrophoreses a clear band (285bp) was detected using TCDD-treated sample, while nothing with control samples. To detect transformed AhR-DRE complex, 2 fmol DNAs and 3 ug cytosol proteins were found to be sufficient in the experiment. Compared with gel retardation assay, this new method is more sensitive for monitoring the Ah receptor-enhancer interaction without radioactive pollution.

  3. Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

    Directory of Open Access Journals (Sweden)

    Kathleen Busman-Sahay

    Full Text Available Following antigen recognition, B cell receptor (BCR-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2 is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs. Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

  4. PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-beta signaling during mammary tumorigenesis. (United States)

    Tian, Maozhen; Schiemann, William P


    The molecular mechanisms that enable cyclooxygenase-2 (COX-2) and its mediator prostaglandin E2 (PGE2) to inhibit transforming growth factor-beta (TGF-beta) signaling during mammary tumorigenesis remain unknown. We show here that TGF-beta selectively stimulated the expression of the PGE2 receptor EP2, which increased normal and malignant mammary epithelial cell (MEC) invasion, anchorage-independent growth, and resistance to TGF-beta-induced cytostasis. Mechanistically, elevated EP2 expression in normal MECs inhibited the coupling of TGF-beta to Smad2/3 activation and plasminogen activator inhibitor-1 (PAI1) expression, while EP2 deficiency in these same MECs augmented Smad2/3 activation and PAI expression stimulated by TGF-beta. Along these lines, engineering malignant MECs to lack EP2 expression prevented their growth in soft agar, restored their cytostatic response to TGF-beta, decreased their invasiveness in response to TGF-beta, and potentiated their activation of Smad2/3 and expression of PAI stimulated by TGF-beta. More important, we show that COX-2 or EP2 deficiency both significantly decreased the growth, angiogenesis, and pulmonary metastasis of mammary tumors produced in mice. Collectively, this investigation establishes EP2 as a potent mediator of the anti-TGF-beta activities elicited by COX-2/PGE2 in normal and malignant MECs. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the oncogenic activities of TGF-beta during mammary tumorigenesis.-Tian, M., Schiemann, W. P. PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-beta signaling during mammary tumorigenesis.

  5. PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-β signaling during mammary tumorigenesis (United States)

    Tian, Maozhen; Schiemann, William P.


    The molecular mechanisms that enable cyclooxygenase-2 (COX-2) and its mediator prostaglandin E2 (PGE2) to inhibit transforming growth factor-β (TGF-β) signaling during mammary tumorigenesis remain unknown. We show here that TGF-β selectively stimulated the expression of the PGE2 receptor EP2, which increased normal and malignant mammary epithelial cell (MEC) invasion, anchorage-independent growth, and resistance to TGF-β-induced cytostasis. Mechanistically, elevated EP2 expression in normal MECs inhibited the coupling of TGF-β to Smad2/3 activation and plasminogen activator inhibitor-1 (PAI1) expression, while EP2 deficiency in these same MECs augmented Smad2/3 activation and PAI expression stimulated by TGF-β. Along these lines, engineering malignant MECs to lack EP2 expression prevented their growth in soft agar, restored their cytostatic response to TGF-β, decreased their invasiveness in response to TGF-β, and potentiated their activation of Smad2/3 and expression of PAI stimulated by TGF-β. More important, we show that COX-2 or EP2 deficiency both significantly decreased the growth, angiogenesis, and pulmonary metastasis of mammary tumors produced in mice. Collectively, this investigation establishes EP2 as a potent mediator of the anti-TGF-β activities elicited by COX-2/PGE2 in normal and malignant MECs. Our findings also suggest that pharmacological targeting of EP2 receptors may provide new inroads to antagonize the oncogenic activities of TGF-β during mammary tumorigenesis.—Tian, M., Schiemann, W. P. PGE2 receptor EP2 mediates the antagonistic effect of COX-2 on TGF-β signaling during mammary tumorigenesis. PMID:19897661

  6. Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway

    Directory of Open Access Journals (Sweden)

    Issa P Bagayogo


    Full Text Available A number of studies suggest that OLGs (oligodendrocytes), the myelinating cells of the central nervous system, are also a source of trophic molecules, such as neurotrophins that may influence survival of proximate neurons. What is less clear is how the release of these molecules may be regulated. The present study investigated the effects of BDNF (brain-derived neurotrophic factor) derived from cortical OLGs on proximate neurons, as well as regulatory mechanisms mediating BDNF release. Initial work determined that BDNF derived from cortical OLGs increased the numbers of VGLUT1 (vesicular glutamate transporter 1)-positive glutamatergic cortical neurons. Furthermore, glutamate acting through metabotropic, and not AMPA/kainate or NMDA (N-methyl-d-aspartate), receptors increased BDNF release. The PLC (phospholipase C) pathway is a key mediator of metabotropic actions to release BDNF in astrocytes and neurons. Treatment of OLGs with the PLC activator m-3M3FBS [N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide] induced robust release of BDNF. Moreover, release elicited by the metabotropic receptor agonist ACPD [trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] was inhibited by the PLC antagonist U73122, the IP3 (inositol triphosphate 3) receptor inhibitor 2-APB (2-aminoethoxydiphenylborane) and the intracellular calcium chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. Taken together, these results suggest that OLG lineage cells release BDNF, a molecule trophic for proximate neurons. BDNF release is regulated by glutamate acting through mGluRs (metabotropic glutamate receptors) and the PLC pathway. Thus glutamate and BDNF may be molecules that support neuron–OLG interactions in the cortex.

  7. The novel heteromeric bivalent ligand SB9 potently antagonizes P2Y(1) receptor-mediated responses. (United States)

    Lambrecht, G; Ganso, M; Bäumert, H G; Spatz-Kümbel, G; Hildebrandt, C; Braun, K; Mutschler, E


    Effects of 6-[(4,6,8-trisulfo-1-naphthyl)iminocarbonyl-1, 3-(4-methylphenylene)iminocarbonyl-1, 3-phenylene-azo]-pyridoxal-5'-phosphate (SB9), a heterodimeric bivalent ligand consisting of pyridoxal-5'-phosphate and the suramin monomer, were studied on contractions of the rat vas deferens elicited by alpha beta-methylene ATP (alpha beta meATP; mediated by P2X(1)-like receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by adenosine 5'-O-(2-thiodiphosphate) (ADP beta S mediated by P2Y(1)-like receptors), and the degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. SB9 (0.1-10 microM) antagonized contractile responses produced by alpha beta meATP or ADP beta S in a concentration-dependent manner. Schild analysis yielded linear regression lines of unit slope, indicating competitive antagonism. From the rightward shifts of the agonist concentration-response curves pA(2) values of 6.05+/-0.13 (vas deferens) and 6.98+/-0.07 (ileum) were derived. In both preparations, SB9 behaved as a slow onset, slow offset antagonist. Incubation of three oocytes in the presence of ATP produced an increase in inorganic phosphate (P(i)) over a 30-min period, which amounted to 35.1+/-1.9 microM P(i) from 100 microM ATP. SB9 (10-1000 microM) reduced this degradation (pIC(50)=4.33+/-0.10). The results illustrate that SB9 is a high-affinity P2Y(1) receptor antagonist with a remarkable selectivity for P2Y(1) vs. P2X(1) receptors (about 10-fold) and ecto-nucleotidases (447-fold). These properties make it unique among the pyridoxal-5'-phosphate and suramin derivatives reported to date.

  8. Glycine and GABAA receptors mediate tonic and phasic inhibitory processes that contribute to prepulse inhibition in the goldfish startle network

    Directory of Open Access Journals (Sweden)

    Paul C.P. Curtin


    Full Text Available Prepulse inhibition (PPI is understood as an inhibitory process that attenuates sensory flow during early stages (20-1000ms of information processing. Here, we applied in vivo electrophysiology and pharmacology to determine if prepulse inhibition (PPI is mediated by glycine receptors (GlyRs and/or GABAA receptors (GABAARs in the goldfish auditory startle circuit. Specifically, we used selective antagonists to dissect the contributions of target receptors on sound-evoked postsynaptic potentials (PSPs recorded in the neurons that initiate startle, the Mauthner-cells (M-cell. We found that strychnine, a GlyR antagonist, disrupted a fast-activated (5 ms and rapidly (< 50ms decaying (feed-forward inhibitory process that disrupts PPI at 20 ms prepulse/pulse inter-stimulus intervals (ISI. Additionally we observed increases of the evoked postsynaptic potential (PSP peak amplitude (+87.43 ± 21.53%; N=9 and duration (+204 ± 48.91%, N=9. In contrast, treatment with bicuculline, a GABAAR antagonist, caused a general reduction in PPI across all tested ISIs (20-500 ms, essentially eliminating PPI at ISIs from 20-100 ms. Bicuculline also increased PSP peak amplitude (+133.8 ± 10.3%, N=5 and PSP duration (+284.95 ± 65.64%, N=5. Treatment with either antagonist also tonically increased post-synaptic excitability in the M-cells, reflected by an increase in the magnitude of antidromically-evoked action potentials (APs by 15.07 ± 3.21%, N=7 and 16.23 ± 7.08%, N=5 for strychnine and bicuculline, respectively. These results suggest that GABAARs and GlyRs are functionally segregated to short- and longer-lasting sound-evoked (phasic inhibitory processes that contribute to PPI, with the mediation of tonic inhibition by both receptor systems being critical for gain control within the M-cell startle circuit.

  9. Differential requirement of the epidermal growth factor receptor for G protein-mediated activation of transcription factors by lysophosphatidic acid

    Directory of Open Access Journals (Sweden)

    Dent Paul


    Full Text Available Abstract Background The role of the epidermal growth factor receptor (EGFR and other receptor tyrosine kinases (RTKs in provoking biological actions of G protein-coupled receptors (GPCRs has been one of the most disputed subjects in the field of GPCR signal transduction. The purpose of the current study is to identify EGFR-mediated mechanisms involved in activation of G protein cascades and the downstream transcription factors by lysophosphatidic acid (LPA. Results In ovarian cancer cells highly responsive to LPA, activation of AP-1 by LPA was suppressed by inhibition of EGFR, an effect that could be reversed by co-stimulation of another receptor tyrosine kinase c-Met with hepatocyte growth factor, indicating that LPA-mediated activation of AP-1 requires activity of a RTK, not necessarily EGFR. Induction of AP-1 components by LPA lied downstream of Gi, G12/13, and Gq. Activation of the effectors of Gi, but not Gq or G12/13 was sensitive to inhibition of EGFR. In contrast, LPA stimulated another prominent transcription factor NF-κB via the Gq-PKC pathway in an EGFR-independent manner. Consistent with the importance of Gi-elicited signals in a plethora of biological processes, LPA-induced cytokine production, cell proliferation, migration and invasion require intact EGFR. Conclusions An RTK activity is required for activation of the AP-1 transcription factor and other Gi-dependent cellular responses to LPA. In contrast, activation of G12/13, Gq and Gq-elicited NF-κB by LPA is independent of such an input. These results provide a novel insight into the role of RTK in GPCR signal transduction and biological functions.

  10. Membrane estrogen receptor-α-mediated nongenomic actions of phytoestrogens in GH3/B6/F10 pituitary tumor cells


    Jeng, Yow-Jiun; Kochukov, Mikhail Y.; Watson, Cheryl S


    Background: Estradiol (E2) mediates various intracellular signaling cascades from the plasma membrane via several estrogen receptors (ERs). The pituitary is an estrogen-responsive tissue, and we have previously reported that E2 can activate mitogen-activated protein kinases (MAPKs) such as ERK1/2 and JNK1/2/3 in the membrane ERα (mERα)-enriched GH3/B6/F10 rat pituitary tumor cell line. Phytoestrogens are compounds found in plants and foods such as soybeans, alfalfa sprouts, and red grapes. Th...

  11. Estrogen-Mediated Upregulation of Noxa Is Associated with Cell Cycle Progression in Estrogen Receptor-Positive Breast Cancer Cells



    Noxa is a Bcl-2-homology domain (BH3)-only protein reported to be a proapoptotic member of the Bcl-2 family. Estrogen has been well documented to stimulate cell growth and inhibit apoptosis in estrogen receptor (ER)-positive breast cancer cells. Intriguingly, recent reports have shown that 17β-estradiol (E2) induces Noxa expression, although the mechanisms underlying E2-mediated induction of Noxa and its functional significance are unknown. Using MCF7 human breast cancer cells as an experimen...

  12. Fine tuning inflammation at the front door: macrophage complement receptor 3-mediates phagocytosis and immune suppression for Francisella tularensis.

    Directory of Open Access Journals (Sweden)

    Shipan Dai


    Full Text Available Complement receptor 3 (CR3, CD11b/CD18 is a major macrophage phagocytic receptor. The biochemical pathways through which CR3 regulates immunologic responses have not been fully characterized. Francisella tularensis is a remarkably infectious, facultative intracellular pathogen of macrophages that causes tularemia. Early evasion of the host immune response contributes to the virulence of F. tularensis and CR3 is an important receptor for its phagocytosis. Here we confirm that efficient attachment and uptake of the highly virulent Type A F. tularensis spp. tularensis strain Schu S4 by human monocyte-derived macrophages (hMDMs requires complement C3 opsonization and CR3. However, despite a>40-fold increase in uptake following C3 opsonization, Schu S4 induces limited pro-inflammatory cytokine production compared with non-opsonized Schu S4 and the low virulent F. novicida. This suggests that engagement of CR3 by opsonized Schu S4 contributes specifically to the immune suppression during and shortly following phagocytosis which we demonstrate by CD11b siRNA knockdown in hMDMs. This immune suppression is concomitant with early inhibition of ERK1/2, p38 MAPK and NF-κB activation. Furthermore, TLR2 siRNA knockdown shows that pro-inflammatory cytokine production and MAPK activation in response to non-opsonized Schu S4 depends on TLR2 signaling providing evidence that CR3-TLR2 crosstalk mediates immune suppression for opsonized Schu S4. Deletion of the CD11b cytoplasmic tail reverses the CR3-mediated decrease in ERK and p38 activation during opsonized Schu-S4 infection. The CR3-mediated signaling pathway involved in this immune suppression includes Lyn kinase and Akt activation, and increased MKP-1, which limits TLR2-mediated pro-inflammatory responses. These data indicate that while the highly virulent F. tularensis uses CR3 for efficient uptake, optimal engagement of this receptor down-regulates TLR2-dependent pro-inflammatory responses by inhibiting

  13. In vitro evidence in rainbow trout supporting glucosensing mediated by sweet taste receptor, LXR, and mitochondrial activity in Brockmann bodies, and sweet taste receptor in liver. (United States)

    Otero-Rodiño, Cristina; Velasco, Cristina; Álvarez-Otero, Rosa; López-Patiño, Marcos A; Míguez, Jesús M; Soengas, José L


    We previously obtained evidence in rainbow trout peripheral tissues such as liver and Brockmann bodies (BB) for the presence and response to changes in circulating levels of glucose (induced by intraperitoneal hypoglycaemic and hyperglycaemic treatments) of glucosensing mechanisms others than that mediated by glucokinase (GK). There were based on mitochondrial production of reactive oxygen species (ROS) leading to increased expression of uncoupling protein 2 (UCP2), and sweet taste receptor in liver and BB, and on liver X receptor (LXR) and sodium/glucose co-transporter 1 (SGLT-1) in BB. We aimed in the present study to obtain further in vitro evidence for the presence and functioning of these systems. In a first experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium containing 2, 4, or 8mM d-glucose, and we assessed the response of parameters related to these glucosensing mechanisms. In a second experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium with 8mM d-glucose alone (control) or containing 1mM phloridzin (SGLT-1 antagonist), 20μM genipin (UCP2 inhibitor), 1μM trolox (ROS scavenger), 100μM bezafibrate (T1R3 inhibitor), and 50μM geranyl-geranyl pyrophosphate (LXR inhibitor). The results obtained in both experiments support the presence and functioning of glucosensor mechanisms in liver based on sweet taste receptor whereas in BB the evidence support those based on LXR, mitochondrial activity and sweet taste receptor.

  14. Oxygen/glucose deprivation induces a reduction in synaptic AMPA receptors on hippocampal CA3 neurons mediated by mGluR1 and adenosine A3 receptors. (United States)

    Dennis, Siobhan H; Jaafari, Nadia; Cimarosti, Helena; Hanley, Jonathan G; Henley, Jeremy M; Mellor, Jack R


    Hippocampal CA1 pyramidal neurons are highly sensitive to ischemic damage, whereas neighboring CA3 pyramidal neurons are less susceptible. It is proposed that switching of AMPA receptor (AMPAR) subunits on CA1 neurons during an in vitro model of ischemia, oxygen/glucose deprivation (OGD), leads to an enhanced permeability of AMPARs to Ca(2+), resulting in delayed cell death. However, it is unclear whether the same mechanisms exist in CA3 neurons and whether this underlies the differential sensitivity to ischemia. Here, we investigated the consequences of OGD for AMPAR function in CA3 neurons using electrophysiological recordings in rat hippocampal slices. Following a 15 min OGD protocol, a substantial depression of AMPAR-mediated synaptic transmission was observed at CA3 associational/commissural and mossy fiber synapses but not CA1 Schaffer collateral synapses. The depression of synaptic transmission following OGD was prevented by metabotropic glutamate receptor 1 (mGluR1) or A(3) receptor antagonists, indicating a role for both glutamate and adenosine release. Inhibition of PLC, PKC, or chelation of intracellular Ca(2+) also prevented the depression of synaptic transmission. Inclusion of peptides to interrupt the interaction between GluA2 and PICK1 or dynamin and amphiphysin prevented the depression of transmission, suggesting a dynamin and PICK1-dependent internalization of AMPARs after OGD. We also show that a reduction in surface and total AMPAR protein levels after OGD was prevented by mGluR1 or A(3) receptor antagonists, indicating that AMPARs are degraded following internalization. Thus, we describe a novel mechanism for the removal of AMPARs in CA3 pyramidal neurons following OGD that has the potential to reduce excitotoxicity and promote neuroprotection.

  15. Consolidation of remote fear memories involves Corticotropin-Releasing Hormone (CRH) receptor type 1-mediated enhancement of AMPA receptor GluR1 signaling in the dentate gyrus. (United States)

    Thoeringer, Christoph K; Henes, Kathrin; Eder, Matthias; Dahlhoff, Maik; Wurst, Wolfgang; Holsboer, Florian; Deussing, Jan M; Moosmang, Sven; Wotjak, Carsten T


    Persistent dreadful memories and hyperarousal constitute prominent psychopathological features of posttraumatic stress disorder (PTSD). Here, we used a contextual fear conditioning paradigm to demonstrate that conditional genetic deletion of corticotropin-releasing hormone (CRH) receptor 1 within the limbic forebrain in mice significantly reduced remote, but not recent, associative and non-associative fear memories. Per os treatment with the selective CRHR1 antagonist DMP696 (3 mg/kg) attenuated consolidation of remote fear memories, without affecting their expression and retention. This could be achieved, if DMP696 was administered for 1 week starting as late as 24 h after foot shock. Furthermore, by combining electrophysiological recordings and western blot analyses, we demonstrate a delayed-onset and long-lasting increase in AMPA receptor (AMPAR) GluR1-mediated signaling in the dentate gyrus (DG) of the dorsal hippocampus 1 month after foot shock. These changes were absent from CRHR1-deficient mice and after DMP696 treatment. Inactivation of hippocampal GluR1-containing AMPARs by antisense oligonucleotides or philantotoxin 433 confirmed the behavioral relevance of AMPA-type glutamatergic neurotransmission in maintaining the high levels of remote fear in shocked mice with intact CRHR1 signaling. We conclude that limbic CRHR1 receptors enhance the consolidation of remote fear memories in the first week after foot shock by increasing the expression of Ca(2+)-permeable GluR1-containing AMPARs in the DG. These findings suggest both receptors as rational targets for the prevention and therapy, respectively, of psychopathology associated with exaggerated fear memories, such as PTSD.

  16. Preparation and characterization of folate-poly(ethylene glycol)-grafted-trimethylchitosan for intracellular transport of protein through folate receptor-mediated endocytosis. (United States)

    Zheng, Yu; Song, Xiangrong; Darby, Michael; Liang, Yufeng; He, Ling; Cai, Zheng; Chen, Qiuhong; Bi, Yueqi; Yang, Xiaojuan; Xu, Jiapeng; Li, Yuanbo; Sun, Yiyi; Lee, Robert J; Hou, Shixiang


    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins to specific tumor cells, folate-poly(ethylene glycol)-grafted-trimethylchitosan (folate-PEG-g-TMC) was synthesized. Nano-scaled spherical polyelectrolyte complexes between the folate-PEG-g-TMC and fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) were prepared under suitable weight ratio of copolymer to FITC-BSA by ionic interaction between the positively charged copolymers and the negatively charged FITC-BSA. Intracellular uptake of FITC-BSA was specifically enhanced in SKOV3 cells (folate receptor over-expressing cell line) through folate receptor-mediated endocytosis compared with A549 cells (folate receptor deficient cell line). Folate-PEG-g-TMC shows promise for intracellular transport of negatively charged therapeutic proteins into folate receptor over-expressing tumor cells.

  17. Cannabinoid CB2 receptor-mediated anti-nociception in models of acute and chronic pain. (United States)

    Jhaveri, Maulik D; Sagar, Devi R; Elmes, Steven J R; Kendall, David A; Chapman, Victoria


    The endocannabinoid system consists of cannabinoid CB(1) and CB(2) receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB(1) receptors. Following the recent evidence for CB(2) receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB(2) receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB(2) receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB(2) receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action.

  18. Histamine H1 and endothelin ETB receptors mediate phospholipase D stimulation in rat brain hippocampal slices. (United States)

    Sarri, E; Picatoste, F; Claro, E


    Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans-(1S,3R)-aminocyclopentyl-1,3-dicarboxylic acid (trans-ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [32P]Pi-prelabeled rat brain cortical and hippocampal slices. The accumulation of [32P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans-ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC50 18 microM) was inhibited by the H1 receptor antagonist mepyramine with a Ki constant of 0.7 nM and was resistant to H2 and H3 receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC50 44 nM) was not blocked by BQ-123, a specific antagonist of the ETA receptor. Endothelin-3 and the specific ETB receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC50 values of 16 and 3 nM, respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H1 and ETB receptors, respectively.

  19. Interleukin-6, A Cytokine Critical to Mediation of Inflammation, Autoimmunity and Allograft Rejection: Therapeutic Implications of IL-6 Receptor Blockade. (United States)

    Jordan, Stanley C; Choi, Jua; Kim, Irene; Wu, Gordon; Toyoda, Mieko; Shin, Bonga; Vo, Ashley


    The success of kidney transplants is limited by the lack of robust improvements in long-term survival. It is now recognized that alloimmune responses are responsible for the majority of allograft failures. Development of novel therapies to decrease allosensitization is critical. The lack of new drug development in kidney transplantation necessitated repurposing drugs initially developed in oncology and autoimmunity. Among these is tocilizumab (anti-IL-6 receptor [IL-6R]) which holds promise for modulating multiple immune pathways responsible for allograft injury and loss. Interleukin-6 is a cytokine critical to proinflammatory and immune regulatory cascades. Emerging data have identified important roles for IL-6 in innate immune responses and adaptive immunity. Excessive IL-6 production is associated with activation of T-helper 17 cell and inhibition of regulatory T cell with attendant inflammation. Plasmablast production of IL-6 is critical for initiation of T follicular helper cells and production of high-affinity IgG. Tocilizumab is the first-in-class drug developed to treat diseases mediated by IL-6. Data are emerging from animal and human studies indicating a critical role for IL-6 in mediation of cell-mediated rejection, antibody-mediated rejection, and chronic allograft vasculopathy. This suggests that anti-IL-6/IL-6R blockade could be effective in modifying T- and B-cell responses to allografts. Initial data from our group suggest anti-IL-6R therapy is of value in desensitization and prevention and treatment of antibody-mediated rejection. In addition, human trials have shown benefits in treatment of graft versus host disease in matched or mismatched stem cell transplants. Here, we explore the biology of IL-6/IL-6R interactions and the evidence for an important role of IL-6 in mediating allograft rejection.

  20. Discoidin Domain Receptor 2 Mediates Collagen-Induced Activation of Membrane-Type 1 Matrix Metalloproteinase in Human Fibroblasts. (United States)

    Majkowska, Iwona; Shitomi, Yasuyuki; Ito, Noriko; Gray, Nathanael S; Itoh, Yoshifumi


    Membrane-Type 1 Matrix Metalloproteinase (MT1-MMP) is a membrane-bound MMP that is highly expressed in cells with invading capacity including fibroblasts and invasive cancer cell. A potential physiological stimulus for MT1-MMP expression is fibrillar collagen, and it has been shown that it upregulates both MT1-MMP gene and functions in various cell types. However, the mechanisms of collagen-mediated MT1-MMP activation is not clearly understood. In this study we identified discoidin domain receptor 2 (DDR2) as a crucial receptor that mediates this process in human fibroblasts. Knocking down DDR2, but not β1 integrin subunit, a common subunit for all collagen-binding integrins, inhibited collagen-induced activation of proMMP-2 and upregulation of MT1-MMP at the gene and protein level. Interestingly DDR2 knockdown or pharmacological inhibition of DDR2 also inhibited MT1-MMP-dependent cellular degradation of collagen film, suggesting that cell surface collagen degradation by MT1-MMP involves DDR2-mediated collagen signalling. This DDR2-mediated mechanism is only present in non-transformed mesenchymal cells, as collagen-induced MT1-MMP activation in HT1080 fibrosarcoma cells and MT1-MMP function in MDA-MB231 breast cancer cells were not affected by DDR kinase inhibition. DDR2 activation was found to be noticeably more effective when cells were stimulated by collagen without non-helical telopeptides region compared to intact collagen fibrils. Those data suggest that DDR2 is a microenvironmental sensor that regulates fibroblasts migration in collagen-rich environment.

  1. Histamine H3A receptor-mediated inhibition of noradrenaline release in the mouse brain cortex. (United States)

    Schlicker, E; Behling, A; Lümmen, G; Göthert, M


    Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused with physiological salt solution containing desipramine plus a drug with alpha 2-adrenoceptor antagonist properties, and the effects of histamine receptor ligands on the electrically (0.3 Hz) evoked tritium overflow were studied. The evoked overflow (from slices superfused with phentolamine) was inhibited by histamine (pIC35 6.53), the H3 receptor agonist R-(-)-alpha-methylhistamine (7.47) and its S-(+)-enantiomer (5.82) but not influenced by the H1 receptor agonist 2-(2-thiazolyl)-ethylamine 3.2 mumol/l and the H2 receptor agonist dimaprit 10 mumol/l. The inhibitory effect of histamine was not affected by the H1 receptor antagonist dimetindene 1 mumol/l and the H2 receptor antagonist ranitidine 10 mumol/l. The concentration-response curve of histamine (determined in the presence of rauwolscine) was shifted to the right by the H3 receptor antagonists thioperamide (apparent pA2 8.67), impromidine (7.30) and burimamide (6.82) as well as by dimaprit (6.16). The pA2 values of the four drugs were compared with their affinities for H3A and H3B binding sites in rat brain membranes (West et al. 1990 Mol Pharmacol 38:610); a significant correlation was obtained for the H3A, but not for the H3B sites. The results suggest that noradrenaline release in the mouse brain cortex is inhibited by histamine via H3A receptors and that dimaprit is an H3 receptor antagonist of moderate potency.

  2. Role of Sigma Receptor in Cocaine-Mediated Induction of Glial Fibrillary Acidic Protein: Implications for HAND. (United States)

    Yang, Lu; Yao, Honghong; Chen, Xufeng; Cai, Yu; Callen, Shannon; Buch, Shilpa


    Cocaine abuse has been shown to accelerate the progression of human immunodeficiency virus (HIV)-1-associated neurological disorders (HANDs) partially through increasing neuroinflammatory response mediated by activated astrocytes; however, the detailed molecular mechanism of cocaine-mediated astrocyte activation is unclear. In the current study, we demonstrated increased astrogliosis in the cortical regions of brains from HIV(+) cocaine abusers compared with the HIV(+) group without cocaine abuse. We next sought to explore whether cocaine exposure could result in increased expression of glial fibrillary acidic protein (GFAP), a filament protein critical for astrocyte activation. Exposure of cocaine to astrocytes resulted in rapid translocation of sigma receptor to the plasma membrane with subsequent activation of downstream signaling pathways. Using a pharmacological approach, we provide evidence that cocaine-mediated upregulation of GFAP expression involved activation of mitogen-activated protein kinase (MAPK) signaling with subsequent downstream activation of the early growth response gene 1 (Egr-1). Egr-1 activation, in turn, caused transcriptional regulation of GFAP. Corroboration of these findings in vivo demonstrated increased expression of GFAP in the cortical region of mice treated with cocaine compared with the saline injected controls. A thorough understanding of how cocaine mediates astrogliosis could have implications for the development of therapeutic interventions aimed at HIV-infected cocaine abusers.

  3. Lipid rafts and raft-mediated supramolecular entities in the regulation of CD95 death receptor apoptotic signaling. (United States)

    Gajate, Consuelo; Mollinedo, Faustino


    Membrane lipid rafts are highly ordered membrane domains enriched in cholesterol, sphingolipids and gangliosides that have the property to segregate and concentrate proteins. Lipid and protein composition of lipid rafts differs from that of the surrounding membrane, thus providing sorting platforms and hubs for signal transduction molecules, including CD95 death receptor-mediated signaling. CD95 can be recruited to rafts in a reversible way through S-palmitoylation following activation of cells with its physiological cognate ligand as well as with a wide variety of inducers, including several antitumor drugs through ligand-independent intracellular mechanisms. CD95 translocation to rafts can be modulated pharmacologically, thus becoming a target for the treatment of apoptosis-defective diseases, such as cancer. CD95-mediated signaling largely depends on protein-protein interactions, and the recruitment and concentration of CD95 and distinct downstream apoptotic molecules in membrane raft domains, forming raft-based supramolecular entities that act as hubs for apoptotic signaling molecules, favors the generation and amplification of apoptotic signals. Efficient CD95-mediated apoptosis involves CD95 and raft internalization, as well as the involvement of different subcellular organelles. In this review, we briefly summarize and discuss the involvement of lipid rafts in the regulation of CD95-mediated apoptosis that may provide a new avenue for cancer therapy.

  4. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    Institute of Scientific and Technical Information of China (English)

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song


    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellula