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Sample records for 5-ht3 receptor activation

  1. 5-HT3 Receptors

    PubMed Central

    Thompson, A. J.; Lummis, S. C. R.

    2009-01-01

    The 5-HT3 receptor is a member of the Cys-loop family of ligand-gated ion channels. These receptors are located in both the peripheral and central nervous systems, where functional receptors are constructed from five subunits. These subunits may be the same (homopentameric 5-HT3A receptors) or different (heteropentameric receptors, usually comprising of 5-HT3A and 5-HT3B receptor subunits), with the latter having a number of distinct properties. The 5-HT3 receptor binding site is comprised of six loops from two adjacent subunits, and critical ligand binding amino acids in these loops have been largely identified. There are a range of selective agonists and antagonists for these receptors and the pharmacophore is reasonably well understood. There are also a wide range of compounds that can modulate receptor activity. Studies have suggested many diverse potential disease targets that might be amenable to alleviation by 5-HT3 receptor selective compounds but to date only two applications have been fully realised in the clinic: the treatment of emesis and irritable-bowel syndrome. PMID:17073663

  2. Design, Synthesis, and Structure–Activity Relationships of Highly Potent 5-HT3 Receptor Ligands

    PubMed Central

    2012-01-01

    The 5-HT3 receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-N-methyl-2-(4-methyl-1,4-diazepan-1-yl)quinazolin-4-amine (1) was identified as a 5-HT3R hit fragment. Here we describe the synthesis and structure–activity relationships (SAR) of a series of (iso)quinoline and quinazoline compounds that were synthesized and screened for 5-HT3R affinity using a [3H]granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound 22 showed the highest affinity (pKi > 10) for the 5-HT3 receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT3 ligands and is used for ligand–receptor binding mode prediction using homology modeling and in silico docking approaches. PMID:23006041

  3. Single-Channel Kinetic Analysis for Activation and Desensitization of Homomeric 5-HT3A Receptors

    PubMed Central

    Corradi, Jeremías; Gumilar, Fernanda; Bouzat, Cecilia

    2009-01-01

    Abstract The 5-HT3A receptor is a member of the Cys-loop family of ligand-gated ion channels. To perform kinetic analysis, we mutated the 5-HT3A subunit to obtain a high-conductance form so that single-channel currents can be detected. At all 5-HT concentrations (>0.1 μM), channel activity appears as openings in quick succession that form bursts, which coalesce into clusters. By combining single-channel and macroscopic data, we generated a kinetic model that perfectly describes activation, deactivation, and desensitization. The model shows that full activation arises from receptors with three molecules of agonist bound. It reveals an earlier conformational change of the fully liganded receptor that occurs while the channel is still closed. From this pre-open closed state, the receptor enters into an open-closed cycle involving three open states, which form the cluster whose duration parallels the time constant of desensitization. A similar model lacking the pre-open closed state can describe the data only if the opening rates are fixed to account for the slow activation rate. The application of the model to M4 mutant receptors shows that position 10′ contributes to channel opening and closing rates. Thus, our kinetic model provides a foundation for understanding structural bases of activation and drug action. PMID:19720021

  4. Activation of 5-HT3 receptors leads to altered responses 6 months after MDMA treatment.

    PubMed

    Gyongyosi, Norbert; Balogh, Brigitta; Katai, Zita; Molnar, Eszter; Laufer, Rudolf; Tekes, Kornelia; Bagdy, Gyorgy

    2010-03-01

    The recreational drug "Ecstasy" [3,4-methylenedioxymethamphetamine (MDMA)] has a well-characterised neurotoxic effect on the 5-hydroxytryptamine (5-HT) neurons in animals. Despite intensive studies, the long-term functional consequencies of the 5-HT neurodegeneration remains elusive. The aim of this study was to investigate whether any alteration of 5-hydroxytryptamine-3 (5-HT(3)) receptor functions on the sleep-wake cycle, motor activity, and quantitative EEG could be detected 6 months after a single dose of 15 mg/kg of MDMA. The selective 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 1 mg/kg, i.p.) or vehicle was administered to freely moving rats pre-treated with MDMA (15 mg/kg, i.p.) or vehicle 6 months earlier. Polysomnographic and motor activity recordings were performed. Active wake (AW), passive wake (PW), light slow wave sleep (SWS-1), deep slow wave sleep (SWS-2), and paradoxical sleep were classified. In addition, EEG power spectra were calculated for the second hour after mCPBG treatment for each stage. AW increased and SWS-1 decreased in the second hour after mCPBG treatment in control animals. mCPBG caused significant changes in the EEG power in states with cortical activation (AW, PW, paradoxical sleep). In addition, mCPBG had a biphasic effect on hippocampal theta power in AW with a decrease in 7 Hz and a stage-selective increase in the upper range (8-9 Hz). Effects of mCPBG on the time spent in AW and SWS-1 were eliminated or reduced in MDMA-treated animals. In addition, mCPBG did not increase the upper theta power of AW in rats pre-treated with MDMA. These data suggest long-term changes in 5-HT(3) receptor function after MDMA. PMID:20052506

  5. Impact of intracellular domain flexibility upon properties of activated human 5-HT3 receptors*

    PubMed Central

    Kozuska, J L; Paulsen, I M; Belfield, W J; Martin, I L; Cole, D J; Holt, A; Dunn, S M J

    2014-01-01

    Background and Purpose It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance. Experimental Approach Mutations were introduced into the portal region of the human 5-HT3A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors. Key Results Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain. Conclusions and Implications These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy. Linked Article This article is commented on by Vardy and Kenakin, pp. 1614–1616 of volume 171 issue 7. To view this commentary

  6. The 5-HT3B subunit affects high-potency inhibition of 5-HT3 receptors by morphine

    PubMed Central

    Baptista-Hon, Daniel T; Deeb, Tarek Z; Othman, Nidaa A; Sharp, Douglas; Hales, Tim G

    2012-01-01

    BACKGROUND AND PURPOSE Morphine is an antagonist at 5-HT3A receptors. 5-HT3 and opioid receptors are expressed in many of the same neuronal pathways where they modulate gut motility, pain and reinforcement. There is increasing interest in the 5-HT3B subunit, which confers altered pharmacology to 5-HT3 receptors. We investigated the mechanisms of inhibition by morphine of 5-HT3 receptors and the influence of the 5-HT3B subunit. EXPERIMENTAL APPROACH 5-HT-evoked currents were recorded from voltage-clamped HEK293 cells expressing human 5-HT3A subunits alone or in combination with 5-HT3B subunits. The affinity of morphine for the orthosteric site of 5-HT3A or 5-HT3AB receptors was assessed using radioligand binding with the antagonist [3H]GR65630. KEY RESULTS When pre-applied, morphine potently inhibited 5-HT-evoked currents mediated by 5-HT3A receptors. The 5-HT3B subunit reduced the potency of morphine fourfold and increased the rates of inhibition and recovery. Inhibition by pre-applied morphine was insurmountable by 5-HT, was voltage-independent and occurred through a site outside the second membrane-spanning domain. When applied simultaneously with 5-HT, morphine caused a lower potency, surmountable inhibition of 5-HT3A and 5-HT3AB receptors. Morphine also fully displaced [3H]GR65630 from 5-HT3A and 5-HT3AB receptors with similar potency. CONCLUSIONS AND IMPLICATIONS These findings suggest that morphine has two sites of action, a low-affinity, competitive site and a high-affinity, non-competitive site that is not available when the channel is activated. The affinity of morphine for the latter is reduced by the 5-HT3B subunit. Our results reveal that morphine causes a high-affinity, insurmountable and subunit-dependent inhibition of human 5-HT3 receptors. PMID:21740409

  7. Influence of the 5-HT3A Receptor Gene Polymorphism and Childhood Sexual Trauma on Central Serotonin Activity

    PubMed Central

    Huh, Hyu Jung; Chae, Jeong-Ho

    2015-01-01

    Background Gene-environment interactions are important for understanding alterations in human brain function. The loudness dependence of auditory evoked potential (LDAEP) is known to reflect central serotonergic activity. Single nucleotide polymorphisms (SNPs) in the 5-HT3A serotonin receptor gene are associated with psychiatric disorders. This study aimed to investigate the effect between 5-HT3A receptor gene polymorphisms and childhood sexual trauma on the LDAEP as an electrophysiological marker in healthy subjects. Methods A total of 206 healthy subjects were recruited and evaluated using the childhood trauma questionnaire (CTQ) and hospital anxiety and depression scale (HADS). Peak-to-peak N1/P2 was measured at five stimulus intensities, and the LDAEP was calculated as the linear-regression slope. In addition, the rs1062613 SNPs of 5-HT3A (CC, CT, and TT) were analyzed in healthy subjects. Results There was a significant interaction between scores on the CTQ-sexual abuse subscale and 5-HT3A genotype on the LDAEP. Subjects with the CC polymorphism had a significantly higher LDEAP than T carriers in the sexually abused group. In addition, CC genotype subjects in the sexually abused group showed a significantly higher LDAEP compared with CC genotype subjects in the non-sexually abused group. Conclusions Our findings suggest that people with the CC polymorphism of the 5-HT3A gene have a greater risk of developing mental health problems if they have experienced childhood sexual abuse, possibly due to low central serotonin activity. Conversely, the T polymorphism may be protective against any central serotonergic changes following childhood sexual trauma. PMID:26701104

  8. Serotonin receptor diversity in the human colon: Expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E

    PubMed Central

    Kapeller, Johannes; Möller, Dorothee; Lasitschka, Felix; Autschbach, Frank; Hovius, Ruud; Rappold, Gudrun; Brüss, Michael; Gershon, Michael D.

    2011-01-01

    Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed immunocytochemistry to locate the 5-HT3A as well as the 5-HT3C, 5-HT3D, and 5-HT3E subunits within the human colon. Western blot analysis was used to confirm subunit expression, and RT-PCR was employed to detect transcripts encoding 5-HT3 receptor subunits in microdissected tissue samples. This investigation revealed, for the first time, that 5-HT3C, 5-HT3D, and 5-HT3E subunits are coexpressed with 5-HT3A in cell bodies of myenteric neurons. Furthermore, 5-HT3A and 5-HT3D were found to be expressed in submucosal plexus of the human large intestine. These data provide a strong basis for future studies of the roles that specific 5-HT3 receptor subtypes play in the function of the enteric and central nervous systems and the contribution that specific 5-HT3 receptors make to the pathophysiology of gastrointestinal disorders such as irritable bowel syndrome and dyspepsia. PMID:21192076

  9. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

    PubMed

    Hoffmann, Katrin M; Herbrechter, Robin; Ziemba, Paul M; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  10. Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors

    PubMed Central

    Hoffmann, Katrin M.; Herbrechter, Robin; Ziemba, Paul M.; Lepke, Peter; Beltrán, Leopoldo; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2016-01-01

    Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications. PMID:27524967

  11. Design, synthesis and evaluation of antidepressant activity of novel 2-methoxy 1, 8 naphthyridine 3-carboxamides as 5-HT3 receptor antagonists.

    PubMed

    Mahesh, Radhakrishnan; Dhar, Arghya Kusum; Jindal, Ankur; Bhatt, Shvetank

    2014-05-01

    A series of novel 1,8-naphthyridine-3-carboxamides as 5-HT3 receptor antagonists were synthesized with an intention to explore the antidepressant activity of these compounds. The title carboxamides were designed using ligand-based approach keeping in consideration the structural requirement of the pharmacophore of 5-HT3 receptor antagonists. The compounds were synthesized using appropriate synthetic route from the starting material nicotinamide. 5-HT3 receptor antagonism of all the compounds, which was denoted in the form of pA2 value, was determined in longitudinal muscle myenteric plexus preparation from guinea-pig ileum against 5-HT3 agonist, 2-methyl-5-HT. Compound 8g (2-methoxy-1, 8-naphthyridin-3-yl) (2-methoxy phenyl piperazine-1-yl) methanone was identified as the most active compound, which expressed a pA2 value of 7.67. The antidepressant activity of all the compounds was examined in mice model of forced swim test (FST); importantly, none of the compounds was found to cause any significant changes in the locomotor activity of mice at the tested dose levels. In FST, the compounds with considerably higher pA2 value exhibited promising antidepressant-like activity, whereas compounds with lower pA2 value did not show antidepressant-like activity as compared to the control group. PMID:24330585

  12. Deletion of the 5-HT3A-receptor subunit blunts the induction of cocaine sensitization

    PubMed Central

    Hodge, C. W.; Bratt, A. M.; Kelley, S. P.

    2008-01-01

    Serotonin (5-HT) receptors are classified into seven groups (5-HT1–7), comprising at least 14 structurally and pharmacologically distinct receptor subtypes. Pharma-cological antagonism of ionotropic 5-HT3 receptors has been shown to modulate both behavioral and neuro-chemical aspects of the induction of sensitization to cocaine. It is not known, however, if specific molecular subunits of the 5-HT3 receptor influence the development of cocaine sensitization. To address this question, we studied the effects of acute and chronic intermittent cocaine administration in mice with a targeted deletion of the gene for the 5-HT3A-receptor subunit (5-HT3A −/−). 5-HT3A (−/−) mice showed blunted induction of cocaine-induced locomotor sensitization as compared with wild-type littermate controls. 5-HT3A (−/−) mice did not differ from wild-type littermate controls on measures of basal motor activity or response to acute cocaine treatment. Enhanced locomotor response to saline injection following cocaine sensitization was observed equally in 5-HT3A (−/−) and wild-type mice suggesting similar conditioned effects associated with chronic cocaine treatment. These data show a role for the 5-HT3A-receptor subunit in the induction of behavioral sensitization to cocaine and suggest that the 5-HT3A molecular subunit modulates neurobehavioral adaptations to cocaine, which may underlie aspects of addiction. PMID:17559417

  13. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves.

    PubMed

    Larson, Eric D; Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C; Finger, Thomas E

    2015-12-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. PMID:26631478

  14. 5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor

    PubMed Central

    Newman, Amy S; Batis, Nikolaos; Grafton, Gillian; Caputo, Francesca; Brady, Catherine A; Lambert, Jeremy J; Peters, John A; Gordon, John; Brain, Keith L; Powell, Andrew D; Barnes, Nicholas M

    2013-01-01

    Background and Purpose The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. Experimental Approach 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. Key Results Cl-indole (1–100 μM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (∼twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. Conclusions and Implications The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor. Linked Articles Recent reviews on allosteric modulation can be found at: Kenakin, T (2013). New concepts in pharmacological efficacy at 7TM receptors: IUPHAR Review 2

  15. Fluvoxamine alleviates seizure activity and downregulates hippocampal GAP-43 expression in pentylenetetrazole-kindled mice: role of 5-HT3 receptors.

    PubMed

    Alhaj, Momen W; Zaitone, Sawsan A; Moustafa, Yasser M

    2015-06-01

    Epilepsy has been documented to lead to many changes in the nervous system including cell loss and mossy fiber sprouting. Neuronal loss and aberrant neuroplastic changes in the dentate gyrus of the hippocampus have been identified in the pentylenetetrazole (PTZ) kindling model. Antiseizure activity of selective serotonin reuptake inhibitors has been reported in several studies. In the current study, the protective effect of fluvoxamine against PTZ-kindling was investigated in terms of seizure scores, neuronal loss, and regulation of hippocampal neuroplasticity. Further, the role of 5-HT3 receptors was determined. Kindling was induced by repeated injections of PTZ (35 mg/kg) thrice weekly, for a total of 13 injections. One hundred male albino mice were allocated into 10 groups: (1) saline, (2) PTZ, (3) diazepam (1 mg/kg)+PTZ, (4-6) fluvoxamine (5, 10 or 20 mg/kg)+PTZ, (7) ondansetron+fluvoxamine (20 mg/kg)+PTZ, (8) ondansetron+PTZ group, (9) ondansetron (2 mg/kg, i.p.)+saline, and (10) fluvoxamine (20 mg/kg)+saline. PTZ-kindled mice showed high seizure activity, hippocampal neuronal loss, and expression of growth-associated phosphoprotein (GAP-43) compared with saline-treated mice. Repeated administration of fluvoxamine (20 mg/kg) in PTZ-kindled mice suppressed seizure scores, protected against hippocampal neuronal loss, and downregulated GAP-43 expression, without producing any signs of the 5-HT syndrome in healthy rats. Importantly, pretreatment with a selective 5-HT3 receptor blocker (ondansetron) attenuated the aforementioned effects of fluvoxamine. In conclusion, the ameliorating effect of fluvoxamine on hippocampal neurons and neuroplasticity in PTZ-kindled mice was, at least in part, dependent on enhancement of hippocampal serotoninergic transmission at 5-HT3 receptors. PMID:25590967

  16. The Role of 5-HT3 Receptors in Signaling from Taste Buds to Nerves

    PubMed Central

    Vandenbeuch, Aurelie; Voigt, Anja; Meyerhof, Wolfgang; Kinnamon, Sue C.; Finger, Thomas E.

    2015-01-01

    Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT3A promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT3A mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μm 5-HT and this response is blocked by 1 μm ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μm m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response. SIGNIFICANCE STATEMENT Historically, serotonin (5-hydroxytryptamine; 5-HT) has been described as a candidate neurotransmitter in the gustatory system and recent studies show that type III taste receptor cells release 5-HT in response to various taste stimuli. In the present study, we demonstrate that a subset of gustatory sensory neurons express functional

  17. 5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems

    PubMed Central

    2015-01-01

    Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined. PMID:25951416

  18. Role of central vagal 5-HT3 receptors in gastrointestinal physiology and pathophysiology

    PubMed Central

    Browning, Kirsteen N.

    2015-01-01

    Vagal neurocircuits are vitally important in the co-ordination and modulation of GI reflexes and homeostatic functions. 5-hydroxytryptamine (5-HT; serotonin) is critically important in the regulation of several of these autonomic gastrointestinal (GI) functions including motility, secretion and visceral sensitivity. While several 5-HT receptors are involved in these physiological responses, the ligand-gated 5-HT3 receptor appears intimately involved in gut-brain signaling, particularly via the afferent (sensory) vagus nerve. 5-HT is released from enterochromaffin cells in response to mechanical or chemical stimulation of the GI tract which leads to activation of 5-HT3 receptors on the terminals of vagal afferents. 5-HT3 receptors are also present on the soma of vagal afferent neurons, including GI vagal afferent neurons, where they can be activated by circulating 5-HT. The central terminals of vagal afferents also exhibit 5-HT3 receptors that function to increase glutamatergic synaptic transmission to second order neurons of the nucleus tractus solitarius within the brainstem. While activation of central brainstem 5-HT3 receptors modulates visceral functions, it is still unclear whether central vagal neurons, i.e., nucleus of the tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) neurons themselves also display functional 5-HT3 receptors. Thus, activation of 5-HT3 receptors may modulate the excitability and activity of gastrointestinal vagal afferents at multiple sites and may be involved in several physiological and pathophysiological conditions, including distention- and chemical-evoked vagal reflexes, nausea, and vomiting, as well as visceral hypersensitivity. PMID:26578870

  19. Impaired Social Behavior in 5-HT3A Receptor Knockout Mice

    PubMed Central

    Smit-Rigter, Laura A.; Wadman, Wytse J.; van Hooft, Johannes A.

    2010-01-01

    The 5-HT3 receptor is a ligand-gated ion channel expressed on interneurons throughout the brain. So far, analysis of the 5-HT3A knockout mouse revealed changes in nociceptive processing and a reduction in anxiety related behavior. Recently, it was shown that the 5-HT3 receptor is also expressed on Cajal-Retzius cells which play a key role in cortical development and that knockout mice lacking this receptor showed aberrant growth of the dendritic tree of cortical layer II/III pyramidal neurons. Other mouse models in which serotonergic signaling was disrupted during development showed similar morphological changes in the cortex, and in addition, also deficits in social behavior. Here, we subjected male and female 5-HT3A knockout mice and their non-transgenic littermates to several tests of social behavior. We found that 5-HT3A knockout mice display impaired social communication in the social transmission of food preference task. Interestingly, we showed that in the social interaction test only female 5-HT3A knockout mice spent less time in reciprocal social interaction starting after 5 min of testing. Moreover, we observed differences in preference for social novelty for male and female 5-HT3A knockout mice during the social approach test. However, no changes in olfaction, exploratory activity and anxiety were detected. These results indicate that the 5-HT3A knockout mouse displays impaired social behavior with specific changes in males and females, reminiscent to other mouse models in which serotonergic signaling is disturbed in the developing brain. PMID:21103015

  20. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

    PubMed Central

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  1. Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel.

    PubMed

    Di Maio, Danilo; Chandramouli, Balasubramanian; Brancato, Giuseppe

    2015-01-01

    Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested. PMID:26465896

  2. Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A)

    PubMed Central

    Brown, A M; Hope, A G; Lambert, J J; Peters, J A

    1998-01-01

    A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 5-HT evoked transient inward currents (EC50 = 3.4 μm; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pm) and by the non-selective agents metoclopramide (IC50 = 69 nm), cocaine (IC50 = 459 nm) and (+)-tubocurarine (IC50 = 2.8 μm). 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of −2.2 mV. N-Methyl-d-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV. Reducing [Ca2+]o from 2 to 0.1 mm caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of γ contributes to this phenomenon. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+. PMID:9508827

  3. Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.

    PubMed

    Werner, P; Kawashima, E; Reid, J; Hussy, N; Lundström, K; Buell, G; Humbert, Y; Jones, K A

    1994-10-01

    The structure of the mouse 5-HT3 receptor gene, 5-HT3R-A, is most similar to nicotinic acetylcholine receptor (nAChR) genes, in particular to the gene encoding the neuronal nAChR subunit alpha 7. These genes share among other things the location of three adjacent introns, suggesting that 5-HT3R-A and nAChR genes arose from a common precursor gene. The alternative use of two adjacent splice acceptor sites in intron 8 creates, in addition to the original 5-HT3R-A cDNA (5-HT3R-AL), a shorter isoform (5-HT3R-AS) which lacks six codons in the segment that translates into the major intracellular domain. This splice consensus sequence is not found in human genomic DNA. In mouse, we demonstrate by RNAse protection assay that 5-HT3R-AS mRNA is approximately 5 times more abundant than 5-HT3R-AL mRNA in both neuroblastoma cell lines and neuronal tissues. We used the Semliki Forest virus expression system for electrophysiological characterization of 5-HT3R-AS and 5-HT3R-AL in mammalian cells. No differences in electrophysiological characteristics, such as voltage dependence, desensitization kinetics, or unitary conductance were found between homomeric 5-HT3R-AS and 5-HT3R-AL receptors. Their properties are very similar to those of 5-HT3 receptors in mouse neuroblastoma cell lines. PMID:7854052

  4. Identification of ginsenoside interaction sites in 5-HT3A receptors.

    PubMed

    Lee, Byung-Hwan; Lee, Jun-Ho; Lee, Sang-Mok; Jeong, Sang Min; Yoon, In-Soo; Lee, Joon-Hee; Choi, Sun-Hye; Pyo, Mi Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Jang, Choon-Gon; Lee, Byoung-Cheol; Park, Chul-Seung; Nah, Seung-Yeol

    2007-03-01

    We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem. PMID:17257631

  5. Serotonin 5-HT3 receptors in rat CA1 hippocampal interneurons: functional and molecular characterization

    PubMed Central

    Sudweeks, Sterling N; van Hooft, Johannes A; Yakel, Jerrel L

    2002-01-01

    The molecular makeup of the serotonin 5-HT3 receptor (5-HT3R) channel was investigated in rat hippocampal CA1 interneurons in slices using single-cell RT-PCR and patch-clamp recording techniques. We tested for the expression of the 5-HT3A (both short and long splice variants) and 5-HT3B subunits, as well as the expression of the α4 subunit of the neuronal nicotinic ACh receptors (nAChRs), the latter of which has been shown to co-assemble with the 5-HT3A subunit in heterologous expression systems. Both the 5-HT3A-short and α4-nAChR subunits were expressed in these interneurons, but we could not detect any expression of either the 5-HT3B or the 5-HT3A-long subunits. Furthermore, there was a strong tendency for the 5-HT3A-short and α4-nAChR subunits to be co-expressed in individual interneurons. To assess whether there was any functional evidence for co-assembly between the 5-HT3A-short and α4-nAChR subunits, we used the sulphydryl agent 2-aminoethyl methanethiosulphonate (MTSEA), which has previously been shown to modulate expressed 5-HT3Rs that contain the α4-nAChR subunit. In half of the interneurons examined, MTSEA significantly enhanced the amplitude of the 5-HT3R-mediated responses, which is consistent with the notion that the α4-nAChR subunit co-assembles with the 5-HT3A subunit to form a native heteromeric 5-HT3R channel in rat CA1 hippocampal interneurons in vivo. In addition, the single-channel properties of the 5-HT3R were investigated in outside-out patches. No resolvable single-channel currents were observed. Using non-stationary fluctuation analysis, we obtained an estimate of the single-channel conductance of 4 pS, which is well below that expected for channels containing both the 5-HT3A and 5-HT3B subunits. PMID:12411518

  6. Cholecystokinin release mediated by 5-HT3 receptors in rat cerebral cortex and nucleus accumbens.

    PubMed Central

    Paudice, P.; Raiteri, M.

    1991-01-01

    1. The effects of 5-hydroxytryptamine (5-HT) on the release of cholexystokinin-like immunoreactivity (CCK-LI) were examined in synaptosomes prepared from rat cerebral cortex and nucleus accumbens and depolarized by superfusion with 15 mM KCl. 2. In both areas 5-HT, tested between 0.1 and 100 nM, increased the calcium-dependent, depolarization-evoked CCK-LI release in a concentration-related manner. The concentration-response curves did not differ significantly between the two brain areas (EC50: 0.4 +/- 0.045 nM and 0.48 +/- 0.053 nM, respectively, in cortical and n. accumbens synaptosomes; maximal effect: about 60% at 10 nM 5-HT). 3. The 5-HT1/5-HT2 receptor antagonist methiothepin (300 nM) did not affect the CCK-LI release elicited by 10 nM 5-HT. However, the effects of 10 nM 5-HT were antagonized in a concentration-dependent manner by the 5-HT3 receptor antagonists (3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1-100 nM; IC50: 3.56 +/- 0.42 nM in the cortex and 3.90 +/- 0.50 nM in the n. accumbens) and ondasetron (IC50: 8.15 +/- 0.73 nM in the cerebral cortex). 5-HT (10 nM) was also strongly antagonized by 100 nM 1 alpha H, 3 alpha 5 alpha H-tropan-3-yl-3,5-dichlorobenzoate (MDL 72222) another blocker of the 5-HT3 receptor. Moreover, the 5-HT3 receptor agonist 1-phenylbiguanide (tested in the cerebral cortex between 0.1 and 100 nM) enhanced CCK-LI release in a manner almost identical to that of 5-HT (EC50 = 0.64 +/- 0.071 nM). 4. It is concluded that 5-HT can act as a potent releaser of CCK-LI in rat cerebrocortex and nucleus accumbens through the activation of receptors of the 5-HT3 type situated on the CCK-releasing terminals. This interaction may provide a rationale for the clinical development of both 5-HT3 and CCK receptor antagonists as novel anxiolytic drugs. PMID:1933141

  7. L-type calcium channels contribute to 5-HT3-receptor-evoked CaMKIIα and ERK activation and induction of emesis in the least shrew (Cryptotis parva).

    PubMed

    Hutchinson, Tarun E; Zhong, Weixia; Chebolu, Seetha; Wilson, Sean M; Darmani, Nissar A

    2015-05-15

    Activation of serotonergic 5-HT3 receptors by its selective agonist 2-methyl serotonin (2-Me-5-HT) induces vomiting, which is sensitive to selective antagonists of both 5-HT3 receptors (palonosetron) and L-type calcium channels (LTCC) (amlodipine or nifedipine). Previously we demonstrated that 5-HT3 receptor activation also causes increases in a palonosetron-sensitive manner in: i) intracellular Ca(2+) concentration, ii) attachment of calmodulin (CaM) to 5-HT3 receptor, and iii) phosphorylation of Ca(2+)/calmodulin-dependent protein kinase IIα (CaMKIIα) and extracellular-signal-regulated kinase 1/2 (ERK1/2). Here, we investigate the role of the short-acting LTCC blocker nifedipine on 2-Me-5-HT-evoked intracellular Ca(2+) increase and on downstream intracellular emetic signaling, which have been shown to be coupled with 2-Me-5-HT׳s emetic effects in the least shrew. Using the cell-permeant Ca(2+) indicator fluo-4 AM, here we present evidence for the contribution of Ca(2+) influx through LTCCs (sensitive to nifedipine) in 2-Me-5-HT (1µM) -evoked rise in cytosolic Ca(2+) levels in least shrew brainstem slices. Nifedipine pretreatment (10mg/kg, s.c.) also suppressed 2-Me-5-HT-evoked interaction of 5-HT3 receptors with CaM as well as phosphorylation of CaMKIIα and ERK1/2 in the least shrew brainstem, and 5-HT3 receptors -CaM colocalization in jejunum of the small intestine. In vitro exposure of isolated enterochromaffin cells of the small intestine to 2-Me-5-HT (1µM) caused CaMKIIα phosphorylation, which was also abrogated by nifedipine pretreatment (0.1µM). In addition, pretreatment with the CaMKII inhibitor KN62 (10mg/kg, i.p.) suppressed emesis and also the activation of CaMKIIα, and ERK in brainstem caused by 2-Me-5-HT (5mg/kg, i.p.). This study provides further mechanistic explanation for our published findings that nifedipine can dose-dependently protect shrews from 2-Me-5-HT-induced vomiting. PMID:25748600

  8. 5-HT3 receptor-dependent modulation of respiratory burst frequency, regularity, and episodicity in isolated adult turtle brainstems

    PubMed Central

    Bartman, Michelle E.; Wilkerson, Julia E.R.; Johnson, Stephen M.

    2010-01-01

    To determine the role of central serotonin 5-HT3 receptors in respiratory motor control, respiratory motor bursts were recorded from hypoglossal (XII) nerve rootlets on isolated adult turtle brainstems during bath-application of 5-HT3 receptor agonists and antagonists. mCPBG and PBG (5-HT3 receptor agonists) acutely increased XII burst frequency and regularity, and decreased bursts/episode. Tropisetron and MDL72222 (5-HT3 antagonists) increased bursts/episode, suggesting endogenous 5-HT3 receptor activation modulates burst timing in vitro. Tropisetron blocked all mCPBG effects, and the PBG-induced reduction in bursts/episode. Tropisetron application following mCPBG application did not reverse the long-lasting (2 h) mCPBG-induced decrease in bursts/episode. We conclude that endogenous 5-HT3 receptor activation regulates respiratory frequency, regularity, and episodicity in turtles and may induce a form of respiratory plasticity with the long-lasting changes in respiratory regularity. PMID:20399913

  9. Impact of Lipid Raft Integrity on 5-HT3 Receptor Function and its Modulation by Antidepressants

    PubMed Central

    Nothdurfter, Caroline; Tanasic, Sascha; Di Benedetto, Barbara; Rammes, Gerhard; Wagner, Eva-Maria; Kirmeier, Thomas; Ganal, Vanessa; Kessler, Julia S; Rein, Theo; Holsboer, Florian; Rupprecht, Rainer

    2010-01-01

    Because of the biochemical colocalization of the 5-HT3 receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT3 receptor function and its modulation by antidepressants. Treatment with methyl-β-cyclodextrine (MβCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MβCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT3 receptor were retained. Although both the 5-HT3 receptor and flotillin-1 were predominantly found in raft-like domains in western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT3 receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT3 receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT3 receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel. PMID:20200506

  10. Unraveling mechanisms underlying partial agonism in 5-HT3A receptors.

    PubMed

    Corradi, Jeremías; Bouzat, Cecilia

    2014-12-10

    Partial agonists have emerged as attractive therapeutic molecules. 2-Me-5HT and tryptamine have been defined as partial agonists of 5-HT3 receptors on the basis of macroscopic measurements. Because several mechanisms may limit maximal responses, we took advantage of the high-conductance form of the mouse serotonin type 3A (5-HT3A) receptor to understand their molecular actions. Individual 5-HT-bound receptors activate in long episodes of high open probability, consisting of groups of openings in quick succession. The activation pattern is similar for 2-Me-5HT only at very low concentrations since profound channel blockade takes place within the activating concentration range. In contrast, activation episodes are significantly briefer in the presence of tryptamine. Generation of a full activation scheme reveals that the fully occupied receptor overcomes transitions to closed preopen states (primed states) before opening. Reduced priming explains the partial agonism of tryptamine. In contrast, 2-Me-5HT is not a genuine partial agonist since priming is not dramatically affected and its low apparent efficacy is mainly due to channel blockade. The analysis also shows that the first priming step is the rate-limiting step and partial agonists require an increased number of priming steps for activation. Molecular docking suggests that interactions are similar for 5-HT and 2-Me-5HT but slightly different for tryptamine. Our study contributes to understanding 5-HT3A receptor activation, extends the novel concept of partial agonism within the Cys-loop family, reveals novel aspects of partial agonism, and unmasks molecular actions of classically defined partial agonists. Unraveling mechanisms underlying partial responses has implications in the design of therapeutic compounds. PMID:25505338

  11. Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP

    PubMed Central

    2015-01-01

    Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor. PMID:25648658

  12. Involvement of 5-HT3 receptors in the action of vortioxetine in rat brain: Focus on glutamatergic and GABAergic neurotransmission.

    PubMed

    Riga, Maurizio S; Sánchez, Connie; Celada, Pau; Artigas, Francesc

    2016-09-01

    The antidepressant vortioxetine is a 5-HT3-R, 5-HT7-R and 5-HT1D-R antagonist, 5-HT1B-R partial agonist, 5-HT1A-R agonist, and serotonin (5-HT) transporter (SERT) inhibitor. Vortioxetine occupies all targets at high therapeutic doses and only SERT and 5-HT3-R at low doses. Vortioxetine increases extracellular monoamine concentrations in rat forebrain more than selective serotonin reuptake inhibitors (SSRI) and shows pro-cognitive activity in preclinical models. Given its high affinity for 5-HT3-R (Ki = 3.7 nM), selectively expressed in GABA interneurons, we hypothesized that vortioxetine may disinhibit glutamatergic and monoaminergic neurotransmission following 5-HT3-R blockade. Here we assessed vortioxetine effect on pyramidal neuron activity and extracellular 5-HT concentration using in vivo extracellular recordings of rat medial prefrontal cortex (mPFC) pyramidal neurons and microdialysis in mPFC and ventral hippocampus (vHPC). Vortioxetine, but not escitalopram, increased pyramidal neuron discharge in mPFC. This effect was prevented by SR57227A (5-HT3-R agonist) and was mimicked by ondansetron (5-HT3-R antagonist) and by escitalopram/ondansetron combinations. In microdialysis experiments, ondansetron augmented the 5-HT-enhancing effect of escitalopram in mPFC and vHPC. Local ondansetron in vHPC augmented escitalopram effect, indicating the participation of intrinsic mechanisms. Since 5-HT neurons express GABAB receptors, we examined their putative involvement in controlling 5-HT release after 5-HT3-R blockade. Co-perfusion of baclofen (but not muscimol) reversed the increased 5-HT levels produced by vortioxetine and escitalopram/ondansetron combinations in vHPC. The present results suggest that vortioxetine increases glutamatergic and serotonergic neurotransmission in rat forebrain by blocking 5-HT3 receptors in GABA interneurons. PMID:27106166

  13. High yield and efficient expression and purification of the human 5-HT3A receptor

    PubMed Central

    Wu, Zhong-shan; Cui, Zhi-cheng; Cheng, Hao; Fan, Chen; Melcher, Karsten; Jiang, Yi; Zhang, Cheng-hai; Jiang, Hua-liang; Cong, Yao; Liu, Qian; Xu, H Eric

    2015-01-01

    Aim: To establish a method for efficient expression and purification of the human serotonin type 3A receptor (5-HT3A) that is suitable for structural studies. Methods: Codon-optimized cDNA of human 5-HT3A was inserted into a modified BacMam vector, which contained an IgG leader sequence, an 8×His tag linked with two-Maltose Binding Proteins (MBP), and a TEV protease cleavage site. The BacMam construct was used to generate baculoviruses for expression of 5-HT3A in HEK293F cells. The proteins were solubilized from the membrane with the detergent C12E 9, and purified using MBP affinity chromatography. The affinity tag was removed by TEV protease treatment and immobilized metal ion affinity chromatography. The receptors were further purified by size-exclusion chromatography (SEC). Western blot and SDS-PAGE were used to detect 5-HT3A during purification. The purified receptor was used in crystallization and analyzed with negative stain electron microscopy (EM). Results: The BacMam system yielded 0.5 milligram of the human 5-HT3A receptor per liter of cells. MBP affinity purification resulted in good yields with high purity and homogeneity. SEC profiles indicated that the purified receptors were pentameric. No protein crystals were obtained; however, a reconstructed 3D density map generated from the negative stain EM data fitted well with the mouse 5-HT3A structure. Conclusion: With the BacMam system, robust expression of the human 5-HT3A receptor is obtained, which is monodisperse, therefore enabling 3D reconstruction of an EM map. This method is suitable for high-throughput screening of different constructs, thus facilitating structural and biochemical studies of the 5-HT3A receptor. PMID:26073329

  14. Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

    PubMed Central

    Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

    2012-01-01

    The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

  15. Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors.

    PubMed

    Gasiorek, Agnes; Trattnig, Sarah M; Ahring, Philip K; Kristiansen, Uffe; Frølund, Bente; Frederiksen, Kristen; Jensen, Anders A

    2016-06-15

    We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT3R) agonists sharing little structural similarity with orthosteric 5-HT3R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT3A receptor (5-HT3AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT3AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT3A receptor suggested that the ligand acts through the transmembrane domain of 5-HT3AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT3ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT3ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT3Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT3AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites. PMID:27086281

  16. Open probability of homomeric murine 5-HT3A serotonin receptors depends on subunit occupancy

    PubMed Central

    Mott, David D; Erreger, Kevin; Banke, Tue G; Traynelis, Stephen F

    2001-01-01

    The time course of macroscopic current responses of homomeric murine serotonin 5-HT3A receptors was studied in whole cells and excised membrane patches under voltage clamp in response to rapid application of serotonin. Serotonin activated whole cell currents with an EC50 value for the peak response of 2 μm and a Hill slope of 3.0 (n = 12), suggesting that the binding of at least three agonist molecules is required to open the channel. Homomeric 5-HT3A receptors in excised membrane patches had a slow activation time course (mean ±s.e.m. 10-90 % rise time 12.5 ± 1.6 ms; n = 9 patches) for 100 μm serotonin. The apparent activation rate was estimated by fitting an exponential function to the rising phase of responses to supramaximal serotonin to be 136 s−1. The 5-HT3A receptor response to 100 μm serotonin in outside-out patches (n = 19) and whole cells (n = 41) desensitized with a variable rate that accelerated throughout the experiment. The time course for desensitization was described by two exponential components (for patches τslow 1006 ± 139 ms, amplitude 31 % τfast 176 ± 25 ms, amplitude 69 %). Deactivation of the response following serotonin removal from excised membrane patches (n = 8) and whole cells (n = 29) was described by a dual exponential time course with time constants similar to those for desensitization (for patches τslow 838 ± 217 ms, 55 % amplitude; τfast 213 ± 44 ms, 45 % amplitude). In most patches (6 of 8), the deactivation time course in response to a brief 1-5 ms pulse of serotonin was similar to or slower than desensitization. This suggests that the continued presence of agonist can induce desensitization with a similar or more rapid time course than agonist unbinding. The difference between the time course for deactivation and desensitization was voltage independent over the range -100 to -40 mV in patches (n = 4) and -100 to +50 mV in whole cells (n = 4), suggesting desensitization of these receptors in the presence of

  17. Functional antagonistic properties of clozapine at the 5-HT3 receptor.

    PubMed

    Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

    1996-08-23

    The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile. PMID:8780717

  18. Antidepressants and antipsychotic drugs colocalize with 5-HT3 receptors in raft-like domains.

    PubMed

    Eisensamer, Brigitte; Uhr, Manfred; Meyr, Sabrina; Gimpl, Gerald; Deiml, Tobias; Rammes, Gerhard; Lambert, Jeremy J; Zieglgänsberger, Walter; Holsboer, Florian; Rupprecht, Rainer

    2005-11-01

    Despite different chemical structure and pharmacodynamic signaling pathways, a variety of antidepressants and antipsychotics inhibit ion fluxes through 5-HT3 receptors in a noncompetitive manner with the exception of the known competitive antagonists mirtazapine and clozapine. To further investigate the mechanisms underlying the noncompetitive inhibition of the serotonin-evoked cation current, we quantified the concentrations of different types of antidepressants and antipsychotics in fractions of sucrose flotation gradients isolated from HEK293 (human embryonic kidney 293) cells stably transfected with the 5-HT3A receptor and of N1E-115 neuroblastoma cells in relation to the localization of the 5-HT3 receptor protein within the cell membrane. Western blots revealed a localization of the 5-HT3 receptor protein exclusively in the low buoyant density (LBD) fractions compatible with a localization within raft-like domains. Also, the antidepressants desipramine, fluoxetine, and reboxetine and the antipsychotics fluphenazine, haloperidol, and clozapine were markedly enriched in LBD fractions, whereas no accumulation occurs for mirtazapine, carbamazepine, moclobemide, and risperidone. The concentrations of psychopharmacological drugs within LBD fractions was strongly associated with their inhibitory potency against serotonin-induced cation currents. The noncompetitive antagonism of antidepressants at the 5-HT3 receptor was not conferred by an enhancement of receptor internalization as shown by immunofluorescence studies, assessment of receptor density in clathrin-coated vesicles, and electrophysiological recordings after coexpression of a dominant-negative mutant of dynamin I, which inhibits receptor internalization. In conclusion, enrichment of antidepressants and antipsychotics in raft-like domains within the cell membrane appears to be crucial for their antagonistic effects at ligand-gated ion channels such as 5-HT3 receptors. PMID:16267227

  19. The muscarinic antagonists scopolamine and atropine are competitive antagonists at 5-HT3 receptors.

    PubMed

    Lochner, Martin; Thompson, Andrew J

    2016-09-01

    Scopolamine is a high affinity muscarinic antagonist that is used for the prevention of post-operative nausea and vomiting. 5-HT3 receptor antagonists are used for the same purpose and are structurally related to scopolamine. To examine whether 5-HT3 receptors are affected by scopolamine we examined the effects of this drug on the electrophysiological and ligand binding properties of 5-HT3A receptors expressed in Xenopus oocytes and HEK293 cells, respectively. 5-HT3 receptor-responses were reversibly inhibited by scopolamine with an IC50 of 2.09 μM. Competitive antagonism was shown by Schild plot (pA2 = 5.02) and by competition with the 5-HT3 receptor antagonists [(3)H]granisetron (Ki = 6.76 μM) and G-FL (Ki = 4.90 μM). The related molecule, atropine, similarly inhibited 5-HT evoked responses in oocytes with an IC50 of 1.74 μM, and competed with G-FL with a Ki of 7.94 μM. The reverse experiment revealed that granisetron also competitively bound to muscarinic receptors (Ki = 6.5 μM). In behavioural studies scopolamine is used to block muscarinic receptors and induce a cognitive deficit, and centrally administered concentrations can exceed the IC50 values found here. It is therefore possible that 5-HT3 receptors are also inhibited. Studies that utilise higher concentrations of scopolamine should be mindful of these potential off-target effects. PMID:27108935

  20. 5-HT3 receptors as important mediators of nausea and vomiting due to chemotherapy.

    PubMed

    Navari, Rudolph M

    2015-10-01

    Chemotherapy-induced nausea and vomiting (CINV) is associated with a significant deterioration in quality of life. The emetogenicity of the chemotherapeutic agents, repeated chemotherapy cycles, and patient risk factors significantly influence CINV. The use of a combination of a 5-hydroxytryptamine-3 (5-HT3) receptor antagonist, dexamethasone, and a neurokinin-1 (NK-1) receptor antagonist has significantly improved the control of acute and delayed emesis in single-day chemotherapy. The first generation 5-HT3 receptor antagonists have been very effective in the control of chemotherapy induced emesis in the first 24 h postchemotherapy (acute emesis), but have not been as effective against delayed emesis (24-120 h postchemotherapy). Palonosetron, a second generation 5-HT3 receptor antagonist with a different half-life, a different binding capacity, and a different mechanism of action than the first generation 5-HT3 receptor antagonists appears to be the most effective agent in its class. Despite the control of emesis, nausea has not been well controlled by current agents. Olanzapine, a FDA approved antipsychotic that blocks multiple neurotransmitters: dopamine at D1, D2, D3, D4 brain receptors, serotonin at 5-HT2a, 5-HT2c, 5-HT3, 5-HT6 receptors, catecholamines at alpha1 adrenergic receptors, acetylcholine at muscarinic receptors, and histamine at H1 receptors, has emerged in recent trials as an effective preventative agent for chemotherapy-induced emesis and nausea, as well as a very effective agent for the treatment of breakthrough emesis and nausea. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers. PMID:25838122

  1. Effects of metformin on intestinal 5-hydroxytryptamine (5-HT) release and on 5-HT3 receptors.

    PubMed

    Cubeddu, L X; Bönisch, H; Göthert, M; Molderings, G; Racké, K; Ramadori, G; Miller, K J; Schwörer, H

    2000-01-01

    Nearly 30% of patients treated with metformin experience gastrointestinal side effects. Since release of 5-hydroxytryptamine (5-HT) from the intestine is associated with nausea, vomiting, and diarrhea, we examined whether metformin induces 5-HT release from the intestinal mucosa. In 40% of tissue biopsy specimens of human duodenal mucosa, metformin (1, 10, and 30 microM) caused an increase in 5-HT outflow by 35, 70, and 98%, respectively. Peak increases in 5-HT outflow were observed after 10-15 min exposure to metformin, returning to baseline levels after 25 min. Tetrodotoxin (1 microM) reduced by about 50% the metformin-evoked increase in 5-HT outflow (P<0.05). Metformin-evoked release was not affected by scopolamine + hexamethonium, propranolol, the 5-HT3 receptor antagonist dolasetron, naloxone, or the NK1 receptor antagonist L703606. In the presence of tetrodotoxin (1 microM), somatostatin (1 microM) further reduced metformin-induced 5-HT release by 15-20%. In view of the 5-HT releasing effects of selective 5-HT3 receptor agonists to which metformin (N-N-dimethylbiguanide) is structurally related, we investigated whether metformin directly interacts with 5-HT3 receptors. Receptor binding (inhibition of [3H]-GR65630 binding) and agonist effects (stimulation of [14C]-guanidinium influx) at 5-HT3 receptors were studied in murine neuroblastoma N1E-115 cells, which express functional 5-HT3 receptors. Metformin up to 0.3 mM failed to inhibit [3H]-GR65630 binding and to modify displacement of [3H]-GR65630 binding induced by 5-HT. 5-HT (3 microM) stimulated the influx of [14C]-guanidinium in intact N1E-115 cells. Metformin up to 1 mM failed to modify basal influx, 5-HT-induced influx, and 5-HT+ substance P-induced influx of [14C]-guanidinium. Our results indicate that metformin induces 5-HT3 receptor-independent release of 5-HT from human duodenal mucosa via neuronal and non-neuronal mechanisms. Part of the gastrointestinal side effects observed during treatment with

  2. Novel and selective partial agonists of 5-HT3 receptors. 2. Synthesis and biological evaluation of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines.

    PubMed

    Prunier, H; Rault, S; Lancelot, J C; Robba, M; Renard, P; Delagrange, P; Pfeiffer, B; Caignard, D H; Misslin, R; Guardiola-Lemaitre, B; Hamon, M

    1997-06-01

    In continuation of our previous work on piperazinopyrrolothienopyrazine derivatives, three series of piperazinopyridopyrrolopyrazines, piperazinopyrroloquinoxalines, and piperazinopyridopyrroloquinoxalines were prepared and evaluated as 5-HT3 receptor ligands. The chemical modifications performed within these new series led to structure-activity relationships regarding both high affinity and selectivity for the 5-HT3 receptors that are in agreement with those established previously for the pyrrolothienopyrazine series. The best compound (8a) obtained in these new series is in the picomolar range of affinity for 5-HT3 receptors with a selectivity higher than 10(6). Four of the high-affinity 5-HT3 ligands (8a, 15a,b, and 16d) were selected in both the pyridopyrrolopyrazine and the pyrroloquinoxaline series and were characterized in vitro and in vivo as agonists or partial agonists. Compound 8a was also evaluated in the light/dark test where it showed potential anxiolytic-like activity at very low doses per os. PMID:9191957

  3. Anti-inflammatory effect of ondansetron through 5-HT3 receptors on TNBS-induced colitis in rat

    PubMed Central

    Motavallian-Naeini, Azadeh; Minaiyan, Mohsen; Rabbani, Mohammad; Mahzuni, Parvin

    2012-01-01

    Inflammatory bowel disease (IBD) is a chronic inflammatory disorder of the intestinal tract whose etiology has not yet been fully elucidated. Available medicines for treatment of IBD are not universally effective and result in marked deleterious effects. This challenge has thus heightened the need for research in order to adopt new therapeutic approaches for the treatment of IBD. 5-HT3 receptor antagonists have shown analgesic and anti-inflammatory properties in vitro and in vivo. Our aim was to investigate the effect of ondansetron, 5-HT3 receptor antagonist, in an immune-based animal model of IBD, trinitrobenzene sulfonic acid (TNBS)-induced rat colitis and probable involvement of 5-HT3 receptors. Two hours after induction of colitis (instillation of 50 mg/kg of TNBS dissolved in 0.25 ml of ethanol 50 % v/v) to male Wistar rats, ondansetron (2 mg/kg), dexamethasone (1 mg/kg), meta-chlorophenylbiguanide (mCPBG, 5 mg/kg), a 5-HT3 receptor agonist, or ondansetron + mCPBG were administrated intraperitoneally (ip) and continued daily for six days. The animals were sacrificed and distal colons were assessed macroscopically, histologically and biochemically [myeloperoxidase (MPO), tumor necrosis factor-alpha, interleukin-6 and interleukin-1 beta]. Ondansetron and dexamethasone resulted in a decrease in macroscopic and microscopic colonic damage significantly. In addition a dramatic reduction in MPO activity and colonic levels of inflammatory cytokines were seen. The protective effects of ondansetron were antagonized by concurrent administration of mCPBG. Our data suggests that the beneficial effects of ondansetron in TNBS-induced colitis could be mediated by 5-HT3 receptors. PMID:27350767

  4. The 5-HT3 receptor is essential for exercise-induced hippocampal neurogenesis and antidepressant effects.

    PubMed

    Kondo, M; Nakamura, Y; Ishida, Y; Shimada, S

    2015-11-01

    Exercise has a variety of beneficial effects on brain structure and function, such as hippocampal neurogenesis, mood and memory. Previous studies have shown that exercise enhances hippocampal neurogenesis, induces antidepressant effects and improves learning behavior. Brain serotonin (5-hydroxytryptamine, 5-HT) levels increase following exercise, and the 5-HT system has been suggested to have an important role in these exercise-induced neuronal effects. However, the precise mechanism remains unclear. In this study, analysis of the 5-HT type 3A receptor subunit-deficient (htr3a(-/-)) mice revealed that lack of the 5-HT type 3 (5-HT3) receptor resulted in loss of exercise-induced hippocampal neurogenesis and antidepressant effects, but not of learning enhancement. Furthermore, stimulation of the 5-HT3 receptor promoted neurogenesis. These findings demonstrate that the 5-HT3 receptor is the critical target of 5-HT action in the brain following exercise, and is indispensable for hippocampal neurogenesis and antidepressant effects induced by exercise. This is the first report of a pivotal 5-HT receptor subtype that has a fundamental role in exercise-induced morphological changes and psychological effects. PMID:25403840

  5. Modulation of dopamine transmission by 5HT2C and 5HT3 receptors: a role in the antidepressant response.

    PubMed

    Dremencov, Eliyahu; Weizmann, Yifat; Kinor, Noa; Gispan-Herman, Iris; Yadid, Gal

    2006-02-01

    Dopaminergic mesolimbic and mesocortical systems are fundamental in hedonia and motivation. Therefore their regulation should be central in understanding depression treatment. This review highlights the dopaminergic activity in relation to depressive behavior and suggests two putative receptors as potential targets for research and development of future antidepressants. In this article we review data that describe the role of serotonin in regulating dopamine release, via 5HT2C and 5HT3 receptors. This action of serotonin appears to be linked to depressive-like behavior and to onset of behavioral effects of antidepressants in an animal model of depression. We suggest that drugs or strategies that decrease 5HT2C and increase 5HT3 receptor-mediated dopamine release in the limbic areas of the brain may provide a fast onset of therapeutic effect. Clinical and basic research data supporting this hypothesis are discussed. PMID:16475958

  6. The function of 5-HT3 receptors on colonic transit in rats.

    PubMed

    Haga, K; Asano, K; Fukuda, T; Kobayakawa, T

    1995-12-01

    The function of serotonin (5-HT)3 receptors on colonic transit was investigated in unanesthetized rats. The colonic transit was accelerated by 5-HT (10 mg/kg, s.c.), 2-methyl-5-HT (30 mg/kg, s.c.), neostigmine (0.03-0.1 mg/kg, s.c.), corticotropin releasing factor (CRF; 1 microgram intracerebroventricular administration) and restraint stress (for 45 minutes). A potent and selective 5-HT3 receptor antagonist, azasetron (+/-)-N-(1-azabicyclo[2.2.2]oct-3-yl)-6-chloro- 4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide monohydrochloride ; 0.01-10 mg/kg, p.o. inhibited the 5-HT-, CRF- and stress-accelerated colonic transit in a dose-dependent manner. Ondansetron (10 mg/kg, p.o.) and granisetron (1 mg/kg, p.o) also inhibited the stress-accelerated colonic transit, but azasetron was more effective than these two drugs. Atropine methylbromide (0.1 mg/kg, s.c.) and tetrodotoxin (0.01 mg/kg, s.c.) inhibited the accelerated colonic transit under stress conditions, but methysergide (10 mg/kg, s.c.), SDZ205-557 (10 mg/kg, s.c.), domperidone (30 mg/kg, p.o.), trimebutine (300 mg/kg, p.o.), did not. Azasetron (10 micrograms) administered intracerebroventricularly did not inhibit the stress-induced acceleration. These results suggest that endogenous 5-HT which is released through stress accelerates the colonic transit via the 5-HT3 receptors and finally a cholinergic mechanism. It is considered that azasetron inhibits colonic transit particularly under stress conditions through the blockade of the peripheral 5-HT3 receptors. Azasetron may improve bowel function in stress-related colonic dysfunction like irritable bowel syndrome. PMID:8653566

  7. The 4′lysine in the putative channel lining domain affects desensitization but not the single-channel conductance of recombinant homomeric 5-HT3A receptors

    PubMed Central

    Gunthorpe, Martin J; Peters, John A; Gill, Catherine H; Lambert, Jeremy J; Lummis, Sarah C R

    2000-01-01

    The 5-HT3 receptor is a transmitter-gated ion channel of the Cys-loop superfamily. Uniquely, 5-HT3 receptor subunits (5-HT3A and 5-HT3B) possess a positively charged lysine residue within the putative channel lining M2 domain (4′ position). Using whole cell recording techniques, we examined the role of this residue in receptor function using wild-type (WT) and mutant 5-HT3A receptor subunits of murine origin transiently expressed in human embryonic kidney (HEK 293) cells. WT 5-HT3A receptors mediated rapidly activating currents in response to 5-HT (10–90 % rise time, 103 ms; EC50, 2.34 μm; Hill coefficient, nH, 2.87). The currents rectified inwardly, reversed in sign at a potential of −9 mV and desensitized in the continuous presence of agonist (half-time of desensitization, t1/2, 2.13 s). 5-HT3A receptor subunits in which the 4′lysine was mutated to arginine, glutamine, serine or glycine formed functional receptors. 5-HT EC50 values were approximately 2-fold lower than for WT 5-HT3A receptors, but Hill coefficients, kinetics of current activation, rectification, and reversal potentials were unaltered. Each of the mutants desensitized more slowly than the WT 5-HT3A receptor, with the arginine and glycine mutations exhibiting the greatest effect (5-fold reduction). The rank order of effect was arginine > glycine > serine > glutamine. The single-channel conductance of the WT 5-HT3A receptor, as assessed by fluctuation analysis of macroscopic currents, was 390 fS. A similar value was obtained for the 4′lysine mutant receptors. Thus it appears unlikely that 4′lysine is exposed to the channel lumen. Mutation of residues immediately adjacent to 4′lysine to glutamate or lysine resulted in lack of receptor expression or function. We conclude that 4′lysine does not form part of the channel lining, but may play an important role in 5-HT3 receptor desensitization. PMID:10639097

  8. The action of SDZ 205,557 at 5-hydroxytryptamine (5-HT3 and 5-HT4) receptors.

    PubMed Central

    Eglen, R. M.; Alvarez, R.; Johnson, L. G.; Leung, E.; Wong, E. H.

    1993-01-01

    1. The interaction of the novel antagonist, SDZ 205,557 (2-methoxy-4-amino-5-chloro benzoic acid 2-(diethylamino) ethyl ester), at 5-HT3 and 5-HT4 receptors has been assessed in vitro and in vivo. 2. In guinea-pig hippocampus and in the presence of 0.4 microM 5-carboxamidotryptamine, 5-HT4-mediated stimulation of adenylyl cyclase was competitively antagonized by SDZ 205,557, with a pA2 value of 7.5, and a Schild slope of 0.81. In rat carbachol-contracted oesophagus, 5-HT4-receptor mediated relaxations were surmountably antagonized by SDZ 205,557 with a similar pA2 value (7.3). This value was agonist-independent with the exception of (R)-zacopride, against which a significantly lower value (6.4) was observed. 3. In functional studies of 5-HT3 receptors, SDZ 205,557 exhibited an affinity of 6.2 in guinea-pig ileum compared with 6.9 at binding sites labelled by [3H]-quipazine in NG108-15 cells. In the anaesthetized, vagotomized micropig, SDZ 205,557 produced only a transient blockade of 5-HT4-mediated tachycardia. This contrasted with tropisetron, which was active for over 60 min after administration. The half-lives for the inhibitory responses of SDZ 205,557 and tropisetron were 23 and 116 min, respectively. 4. In conclusion, SDZ 205,557 has similar affinity for 5-HT3 and 5-HT4 receptors. The apparent selectivity observed in guinea-pig is due to the atypical nature of the 5-HT3 receptor in this species. The short duration of action of this novel antagonist may complicate its use in vivo. SDZ 205,557 should, therefore, be used with appropriate caution in studies defining the 5-HT4 receptor. PMID:8448587

  9. Theoretical evaluation of antiemetic effects of 5-HT3 receptor antagonists for prevention of vomiting induced by cisplatin.

    PubMed

    Nakamura, Hironori; Yokoyama, Haruko; Takayanagi, Risa; Yoshimoto, Koichi; Nakajima, Akihiro; Okuyama, Kiyoshi; Iwase, Osamu; Yamada, Yasuhiko

    2015-03-01

    5-HT(3) receptor antagonists are widely used as antiemetic agents in clinical setting, of which palonosetron, with a long elimination half life (t(1/2)), has recently become available. It is important to evaluate the concentration of serotonin when investigating the antiemetic effects of 5-HT(3) receptor antagonists, as those effects are not based solely on the t(1/2) value. We theoretically evaluated the antiemetic effects of three 5-HT(3) receptor antagonists (granisetron, azasetron, palonosetron) on cisplatin-induced nausea and vomiting by estimating the time course of the 5-HT(3) receptor occupancy of serotonin. We estimated the 5-HT(3) receptor occupancy of serotonin in the small intestine, based on the time course of plasma concentration of each 5-HT(3) receptor antagonist and the time course of concentration of serotonin near the 5-HT(3) receptor in the small intestine after administration of cisplatin. The antiemetic effect of each 5-HT(3) receptor antagonist was evaluated based on the normal level of 5-HT(3) receptor occupancy of serotonin. Our results suggest that an adequate antiemetic effect will be provided when a dose of 75 mg/m(2) of cisplatin is given to patients along with any single administration of granisetron, azasetron, or palonosetron at a usual dose. On the other hand, the 5-HT(3) receptor occupancy of serotonin was found to be significantly lower than normal for several days after administration of palonosetron, as compared to granisetron and azasetron, indicating that constipation may be induced. Our results show that granisetron, azasetron, and palonosetron each have an adequate antiemetic effect after administration of 75 mg/m(2) of cisplatin. PMID:24470169

  10. Identification of Glycyrrhiza as the rikkunshito constituent with the highest antagonistic potential on heterologously expressed 5-HT3A receptors due to the action of flavonoids

    PubMed Central

    Herbrechter, Robin; Ziemba, Paul M.; Hoffmann, Katrin M.; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-01-01

    The traditional Japanese phytomedicine rikkunshito is traditionally used for the treatment of gastrointestinal motility disorders, cachexia and nausea. These effects indicate 5-HT3 receptor antagonism, due to the involvement of these receptors in such pathophysiological processes. E.g., setrons, specific 5-HT3 receptor antagonists are the strongest antiemetics, developed so far. Therefore, the antagonistic effects of the eight rikkunshito constituents at heterologously expressed 5-HT3Areceptors were analyzed using the two-electrode voltage-clamp technique. The results indicate that tinctures from Aurantii, Ginseng, Zingiberis, Atractylodis and Glycyrrhiza inhibited the 5-HT3A receptor response, whereas the tinctures of Poria cocos, Jujubae and Pinellia exhibited no effect. Surprisingly, the strongest antagonism was found for Glycyrrhiza, whereas the Zingiberis tincture, which is considered to be primarily responsible for the effect of rikkunshito, exhibited the weakest antagonism of 5-HT3A receptors. Rikkunshito contains various vanilloids, ginsenosides and flavonoids, a portion of which show an antagonistic effect on 5-HT3 receptors. A screening of the established ingredients of the active rikkunshito constituents and related substances lead to the identification of new antagonists within the class of flavonoids. The flavonoids (-)-liquiritigenin, glabridin and licochalcone A from Glycyrrhiza species were found to be the most effective inhibitors of the 5-HT-induced currents in the screening. The flavonoids (-)-liquiritigenin and hesperetin from Aurantii inhibited the receptor response in a non-competitive manner, whereas glabridin and licochalcone A exhibited a potential competitive antagonism. Furthermore, licochalcone A acts as a partial antagonist of 5-HT3A receptors. Thus, this study reveals new 5-HT3A receptor antagonists with the aid of increasing the comprehension of the complex effects of rikkunshito. PMID:26191003

  11. Fluorophore assisted light inactivation (FALI) of recombinant 5-HT3A receptor constitutive internalization and function

    PubMed Central

    Morton, Russell A.; Luo, Guoxiang; Davis, Margaret I.; Hales, Tim G.; Lovinger, David M.

    2011-01-01

    Fluorescent proteins and molecules are now widely used to tag and visualize proteins resulting in an improved understanding of protein trafficking, localization, and function. In addition, fluorescent tags have also been used to inactivate protein function in a spatially and temporally-defined manner, using a technique known as fluorophore-assisted light inactivation (FALI) or chromophore-assisted light inactivation (CALI). In this study we tagged the serotonin3 A subunit with the α-bungarotoxin binding sequence (BBS) and subsequently labeled 5-HT3A/BBS receptors with fluorescently conjugated α-bungarotoxin in live cells. We show that 5-HT3A/BBS receptors are constitutively internalized in the absence of an agonist and internalization as well as receptor function are inhibited by fluorescence. The fluorescence-induced disruption of function and internalization was reduced with oxygen radical scavengers suggesting the involvement of reactive oxygen species, implicating the FALI process. Furthermore, these data suggest that intense illumination during live-cell microscopy may result in inadvertent FALI and inhibition of protein trafficking. PMID:21338684

  12. On the voltage-dependent Ca2+ block of serotonin 5-HT3 receptors: a critical role of intracellular phosphates

    PubMed Central

    Noam, Yoav; Wadman, Wytse J; van Hooft, Johannes A

    2008-01-01

    Natively expressed serotonin 5-HT3 receptors typically possess a negative-slope conductance region in their I–V curve, due to a voltage-dependent block by external Ca2+ ions. However, in almost all studies performed with heterologously expressed 5-HT3 receptors, this feature was not observed. Here we show that mere addition of ATP to the pipette solution is sufficient to reliably observe a voltage-dependent block in homomeric (h5-HT3A) and heteromeric (h5-HT3AB) receptors expressed in HEK293 cells. A similar block was observed with a plethora of molecules containing a phosphate moiety, thus excluding a role of phosphorylation. A substitution of three arginines in the intracellular vestibule of 5-HT3A with their counterpart residues from the 5-HT3B subunit (RRR-QDA) was previously shown to dramatically increase single channel conductance. We find this mutant to have a linear I–V curve that is unaffected by the presence of ATP, with a fractional Ca2+ current (Pf%) that is reduced (1.8 ± 0.2%) compared to that of the homomeric receptor (4.1 ± 0.2%), and similar to that of the heteromeric form (2.0 ± 0.3%). Moreover, whereas ATP decreased the Pf% of the homomeric receptor, this was not observed with the RRR-QDA mutant. Finally, ATP was found to be critical for voltage-dependent channel block also in hippocampal interneurons that natively express 5-HT3 receptors. Taken together, our results indicate a novel mechanism by which ATP, and similar molecules, modulate 5-HT3 receptors via interactions with the intracellular vestibule of the receptor. PMID:18566001

  13. Functional evidence for the rapid desensitization of 5-HT(3) receptors on vagal afferents mediating the Bezold-Jarisch reflex

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    2000-01-01

    The aim of this study was to determine whether 5-hydroxytryptamine (5-HT)(3) receptors on cardiopulmonary afferents mediating the Bezold-Jarisch reflex (BJR) desensitize upon repeated exposure to selective agonists. BJR-mediated falls in heart rate, diastolic arterial blood pressure and cardiac output elicited by the 5-HT(3)-receptor agonists, phenylbiguanide (100 microg/kg, i.v.) or 2-methyl-5-HT (100 microg/kg, i.v.), progressively diminished upon repeated injection in conscious rats. The BJR responses elicited by 5-HT (40 microg/kg, i.v.) were markedly reduced in rats which had received the above injections of phenylbiguanide or 2-methyl-5-HT whereas the BJR responses elicited by L-S-nitrosocysteine (10 micromol/kg, i.v.) were similar before and after the injections of the 5-HT(3) receptor agonists. These findings suggest that tachyphylaxis to 5-HT(3) receptor agonists may be due to the desensitization of 5-HT(3) receptors on cardiopulmonary afferents rather than the impairment of the central or peripheral processing of the BJR.

  14. Impact of 5-HT3 receptor antagonists on chemotherapy-induced nausea and vomiting: a retrospective cohort study

    PubMed Central

    2012-01-01

    Background 1st generation 5-hydroxytryptamine receptor antagonists (5-HT3 RAs), and palonosetron, a 2nd generation 5-HT3 RA, are indicated for the prevention of chemotherapy (CT)-induced nausea and vomiting (CINV) associated with moderately (MEC) and highly emetogenic CT agents (HEC). This study explores the impact of step therapy policies requiring use of an older 5-HT3 RA before palonosetron on risk of CINV associated with hospital or emergency department (ED) admissions. Methods Patients who received cyclophosphamide post breast cancer (BC) surgery or who were diagnosed with lung cancer on carboplatin (LC-carboplatin) or cisplatin (LC-cisplatin) were selected from PharMetrics’ (IMS LifeLink) claims dataset (2005-2008). Patients were followed for 6 months from initial CT administration for CINV events identified through ICD-9-CM codes. Patients were grouped into those initiated with older, generic 5-HT3 RAs (ondansetron, granisetron, and dolasetron) and those initiated and maintained on palonosetron throughout study follow-up. CINV events and CINV days were analyzed using multivariate regressions controlling for demographic and clinical variables. Results Eligible patients numbered 3,606 in BC, 4,497 in LC-carboplatin and 1,154 in LC-cisplatin cohorts, with 52%, 40%, and 34% in the palonosetron group, respectively. There was no significant difference between the two 5-HT3 RA groups in age or Charlson Comorbidity Index among the two MEC cohorts (BC and LC-carboplatin). Among the LC-cisplatin cohort, palonosetron users were older with more males than the older 5-HT3 RA group (age: 60.1 vs. 61.3; males, 66.9% vs. 56.9%). Compared to the older 5-HT3 RAs, the palonosetron groups incurred 22%-51% fewer 5-HT3 RA pharmacy claims, had fewer patients with CINV events (3.5% vs. 5.5% in BC, 9.5% vs. 12.8% in LC-carboplatin, 16.4% vs. 21.7% in LC-cisplatin), and had lower risk for CINV events (odds ratios 0.62, 0.71, or 0.71, respectively; p < 0.05). The BC and LC

  15. Spinal 5-HT3 receptors mediate descending facilitation and contribute to behavioral hypersensitivity via a reciprocal neuron-glial signaling cascade

    PubMed Central

    2014-01-01

    Background It has been recently recognized that the descending serotonin (5-HT) system from the rostral ventromedial medulla (RVM) in the brainstem and the 5-HT3 receptor subtype in the spinal dorsal horn are involved in enhanced descending pain facilitation after tissue and nerve injury. However, the mechanisms underlying the activation of the 5-HT3 receptor and its contribution to facilitation of pain remain unclear. Results In the present study, activation of spinal 5-HT3 receptors by intrathecal injection of a selective 5-HT3 receptor agonist SR 57227 induced spinal glial hyperactivity, neuronal hyperexcitability and pain hypersensitivity in rats. We found that there was neuron-to-microglia signaling via the chemokine fractalkine, microglia to astrocyte signaling via cytokine IL-18, astrocyte to neuronal signaling by IL-1β, and enhanced activation of NMDA receptors in the spinal dorsal horn. Glial hyperactivation in spinal dorsal horn after hindpaw inflammation was also attenuated by molecular depletion of the descending 5-HT system by intra-RVM Tph-2 shRNA interference. Conclusions These findings offer new insights into the cellular and molecular mechanisms at the spinal level responsible for descending 5-HT-mediated pain facilitation during the development of persistent pain after tissue and nerve injury. New pain therapies should focus on prime targets of descending facilitation-induced glial involvement, and in particular the blocking of intercellular signaling transduction between neurons and glia. PMID:24913307

  16. The analgesic effect of clonixine is not mediated by 5-HT3 subtype receptors.

    PubMed

    Paeile, C; Bustamante, S E; Sierralta, F; Bustamante, D; Miranda, H F

    1995-10-01

    1. The analgesic effect of clonixinate of L-lysine (Clx) in the nociceptive C-fiber reflex in rat and in the writhing test in mice is reported. 2. Clx was administered by three routes, i.v., i.t. and i.c.v., inducing a dose-dependent antinociception. 3. The antinociceptive effect of Clx was 40-45% with respect to the control integration values in the nociceptive C-fiber reflex method. 4. The writhing test yielded ED50 values (mg/kg) of 12.0 +/- 1.3 (i.p.), 1.8 +/- 0.2 (i.t.) and 0.9 +/- 0.1 (i.c.v.) for Clx administration. 5. Ondansetron was not able to antagonize the antinociception response of Clx in the algesiometric tests used. 6. Chlorophenilbiguanide did not produce any significative change in the analgesic effect of Clx in the nociceptive C-fiber reflex method. 7. It is suggested that the mechanism of action of the central analgesia of Clx is not mediated by 5-HT3 subtype receptors. PMID:7590133

  17. Interaction of pyridostigmine with the 5-HT(3) receptor antagonist ondansetron in guinea pigs

    SciTech Connect

    Capacio, B.R.; Byers, C.E.; Matthews, R.L.; Anderson, D.R.; Anders, J.C.

    1993-05-13

    Serotonin receptor subtype three (5HT3) antagonists, such as the drug ondansetron (OND), have been developed as effective anti-emetic compounds. The purpose of this study was to assess the drug interactions of OND (10, 20 and 30 mg/kg) with the organophosphorus pretreatment compound pyridostigmine (PYR; 0.94 mg/kg) after simultaneous oral administration to guinea pigs. Compatibility was assessed by determining (1) OND pharmacokinetics in the absence (Phase 1) and presence (Phase 2) of pyridostigmine (PYR) and (2) PYR-induced acetylcholinesterase (AChE) inhibition kinetics in the absence (Phase 1) and the presence (Phase 2) of OND. AChE inhibition was examined because it has been shown to be an indicator of PYR efficacy against OP-induced lethality. The pharmacokinetics of OND alone and in the presence of PYR were linear and best described by a one-compartment model with first-order absorption and elimination rate kinetics. For OND 30 mg/kg the K10 was found to be significantly smaller in Phase 2 than Phase 1 (p < 0.05).

  18. Intrathecal 5-methoxy-N,N-dimethyltryptamine in mice modulates 5-HT1 and 5-HT3 receptors.

    PubMed

    Alhaider, A A; Hamon, M; Wilcox, G L

    1993-11-01

    The antinociceptive effects of intrathecally administered 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a potent 5-HT receptor agonist, were studied in three behavioral tests in mice: the tail-flick test and the intrathecal substance P and N-methyl-D-aspartic acid (NMDA) assays. Intrathecal administration of 5-MeO-DMT (4.6-92 nmol/mouse) produced a significant prolongation of the tail-flick latency. This action was blocked by 5-HT3 and gamma-aminobutyric acidA (GABAA) receptor antagonists but not by 5-HT2, 5-HT1A, 5-HT1B or 5-HT1S receptor antagonists. Binding studies indicated that 5-MeO-DMT had very low affinity for 5-HT3 receptors. 5-MeO-DMT inhibited biting behavior while increasing scratching behavior induced by intrathecally administered substance P. The inhibition of biting behavior was antagonized by intrathecal co-administration of 5-HT1B and GABAA receptor antagonists while 5-HT1A, 5-HT1S, 5-HT2 and 5-HT3 receptor antagonists had no effect. 5-MeO-DMT-enhanced scratching behavior was inhibited by all the antagonists used except ketanserin and bicuculline, suggesting the involvement of 5-HT1A, 5-HT1B, 5-HT1S, 5-HT3 and GABAA receptors. NMDA-induced biting behavior was inhibited by 5-MeO-DMT pretreatment; this action was antagonized by 5-HT1B, 5-HT3 and GABAA receptor antagonists. The involvement of these receptors in 5-MeO-DMT action suggests that it may promote release of 5-HT (5-hydroxytryptamine, serotonin). PMID:7507056

  19. Differential and additive suppressive effects of 5-HT3 (palonosetron)- and NK1 (netupitant)-receptor antagonists on cisplatin-induced vomiting and ERK1/2, PKA and PKC activation.

    PubMed

    Darmani, Nissar A; Zhong, Weixia; Chebolu, Seetha; Mercadante, Frank

    2015-04-01

    To better understand the anti-emetic profile of the 5-HT3 (palonosetron)- and the tachykinin NK1 (netupitant) -receptor antagonists, either alone or in combination, we evaluated the effects of palonosetron and/or netupitant pretreatment on cisplatin-evoked vomiting and changes in the phosphorylation of brainstem kinases such as the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), protein kinase C alpha/beta (PKCα/β), and protein kinase A (PKA) in the least shrew. Our results demonstrate that cisplatin (10mg/kg, i.p.) causes emesis in the least shrew over 40h with respective peak early- and delayed-phases occurring at 1 - 2 and 32 - 34h post-injection. During the early phase (0 - 16h post cisplatin), palonosetron (0.1mg/kg, s.c.) significantly protected shrews from vomiting with a near complete suppression of vomit frequency. Palonosetron also significantly protected shrews from vomiting during the delayed phase (27 - 40h post cisplatin), but the reduction in mean vomit frequency failed to achieve significance. On the other hand, netupitant (5mg/kg, i.p.) totally abolished vomiting during the delayed phase, and tended to suppress the mean vomit frequency during the acute phase. The combined treatment protected shrews almost completely from vomiting during both phases. Brainstem pERK1/2 levels were significantly elevated at all time-points except at 40h post-cisplatin administration. PKA phosphorylation tended to be elevated throughout the delayed phase, but a significant increase only occurred at 33h. Brainstem pPKCα/β levels were enhanced during acute-phase with a significant elevation at 2h. Palonosetron, netupitant or their combination had no effect on elevated pERK1/2 levels during acute phase, but the combination reversed ERK1/2 phosphorylation at 33h post-cisplatin treatment. In addition, only the combined regimen prevented the cisplatin-induced PKCα/β phosphorylation observed at the acute phase. On the other hand, palonosetron and

  20. Involvement of 5HT3 Receptors in Anti-Inflammatory Effects of Tropisetron on Experimental TNBS-Induced Colitis in Rat

    PubMed Central

    Motavallian, Azadeh; Minaiyan, Mohsen; Rabbani, Mohammad; Andalib, Sasan; Mahzouni, Parvin

    2013-01-01

    Introduction: There is a pressing need for research leading to the development of new effective drugs with lower side effects and more efficacy for treating inflammatory bowel disease (IBD). The analgesic and anti-inflammatory properties of 5-Hydroxytryptamine (5-HT)-3 receptor antagonists have been shown in in vivo and in vitro studies. The present study was designed to investigate the effects of tropisetron, a 5-HT3 receptor antagonist, on an immune-based animal model of IBD. Methods: In the present study, the trinitrobenzenesulfonic acid (TNBS) model of colitis in the rat was used. Two hours after induction of colitis in rats, tropisetron (2 mg/kg), dexamethasone (1 mg/kg), meta-chlorophenylbiguanide (mCPBG, 5 mg/kg), a 5-HT3 receptor agonist, or tropisetron + mCPBG were intraperitoneally (i.p.) administrated for 6 days. Animals were then sacrificed; macroscopic, histological, biochemical (myeloperoxidase [MPO]) assessments and ELISA test (tumor necrosis factor-alpha, interleukin-6 and interleukin-1 beta) were performed on distal colon samples. Results: Tropisetron or dexamethasone treatment significantly reduced macroscopic and microscopic colonic damages. In addition, a significant reduction in MPO activity and colonic levels of inflammatory cytokines was seen. The beneficial effects of tropisetron were antagonized by concurrent administration of mCPBG. Conclusion: The present study indicates that the protective effects of tropisetron on TNBS-induced colitis can be mediated by 5-HT3 receptors. PMID:24455480

  1. Is all radiation-induced emesis ameliorated by 5-HT3 receptor antagonists. (Reannouncement with new availability information)

    SciTech Connect

    Rabin, B.M.; King, G.L.

    1992-12-31

    Exposing ferrets to gamma rays or X-rays produces vomiting that can be attenuated by 5-HT3 receptor antagonists and by subdiaphragmatic vagotomy. The present experiments evaluated the effectiveness of these treatments on emesis evoked by exposure to other types of radiation, fast neutrons from a nuclear reactor and high-energy protons (200 MeV), which differ in the relative effectiveness with which they produce vomiting. The results indicated that higher doses of 5-HT3 receptor antagonists Eusatron (0.03 and 0.30 mg/kg, s.c.) and Ondansetron (0.10 and 0.30 mg/kg, s.c.) prevented emesis following neutron irradiation. Lower doses of these 5-HT3 receptor antagonists and subdiaphragmatic vagotomy attenuated neutron-induced emesis, increasing the latency and decreasing the severity of the emetic episodes. Ondansetron (0.50 and 1.00 mg/kg, s.c.) completely prevented vomiting following exposure to high-energy protons. The results are interpreted as indicating that similar 5-HT3-dependent mechanisms mediate emesis produced by exposure to different types of radiation, despite differences in their relative effectiveness.

  2. Association of 5-HT3B Receptor Gene Polymorphisms with the Efficacy of Ondansetron for Postoperative Nausea and Vomiting

    PubMed Central

    Kim, Min-Soo; Lee, Jeong-Rim; Choi, Eun-Mi; Kim, Eun Ho

    2015-01-01

    Purpose Postoperative nausea and vomiting (PONV) is a common problem after general anesthesia. Although 5-hydroxytryptamine type 3 (5-HT3) receptor antagonists have significantly reduced PONV, over 35% of patients treated with ondansetron can experience PONV. In this study, we investigated whether the Y129S and -100_-102AAG deletion polymorphisms of the 5-HT3B receptor gene affect the efficacy of ondansetron in preventing PONV. Materials and Methods Two hundred and forty-five adult patients who underwent laparoscopic cholecystectomy were enrolled. Ondansetron 0.1 mg/kg was intravenously administered 30 minutes before the end of surgery. Genomic DNA was prepared from blood samples using a nucleic acid isolation device. Both the Y129S variant and the -100_-102AAG deletion variant were screened for using a single base primer extension assay and a DNA direct sequencing method, respectively. The relationship between genetic polymorphisms and clinical outcomes of ondansetron treatment was investigated. Results Among the 5-HT3B AAG deletion genotypes, the incidence of PONV was higher in patients with the homomutant than with other genotypes during the first 2 hours after surgery (p=0.02). There were no significant differences in the incidence of PONV among genotypes at 2-24 hours after surgery. In the Y129S variants of the 5-HT3B receptor gene, there were no significant differences in the incidence of PONV among genotypes during the first 2 hours and at 2-24 hours after surgery. Conclusion The response to ondansetron for PONV was significantly influenced by the -100_-102AAG deletion polymorphisms of the 5-HT3B gene. Thus, the -100_-102AAG deletion variants may be a pharmacogenetic predictor for responsiveness to ondansetron for PONV. PMID:26256989

  3. 5-Hydroxytryptamine (5-HT) Cellular Sequestration during Chronic Exposure Delays 5-HT3 Receptor Resensitization due to Its Subsequent Release*

    PubMed Central

    Hothersall, J. Daniel; Alexander, Amy; Samson, Andrew J.; Moffat, Christopher; Bollan, Karen A.; Connolly, Christopher N.

    2014-01-01

    The serotonergic synapse is dynamically regulated by serotonin (5-hydroxytryptamine (5-HT)) with elevated levels leading to the down-regulation of the serotonin transporter and a variety of 5-HT receptors, including the 5-HT type-3 (5-HT3) receptors. We report that recombinantly expressed 5-HT3 receptor binding sites are reduced by chronic exposure to 5-HT (IC50 of 154.0 ± 45.7 μm, t½ = 28.6 min). This is confirmed for 5-HT3 receptor-induced contractions in the guinea pig ileum, which are down-regulated after chronic, but not acute, exposure to 5-HT. The loss of receptor function does not involve endocytosis, and surface receptor levels are unaltered. The rate and extent of down-regulation is potentiated by serotonin transporter function (IC50 of 2.3 ± 1.0 μm, t½ = 3.4 min). Interestingly, the level of 5-HT uptake correlates with the extent of down-regulation. Using TX-114 extraction, we find that accumulated 5-HT remains soluble and not membrane-bound. This cytoplasmically sequestered 5-HT is readily releasable from both COS-7 cells and the guinea pig ileum. Moreover, the 5-HT level released is sufficient to prevent recovery from receptor desensitization in the guinea pig ileum. Together, these findings suggest the existence of a novel mechanism of down-regulation where the chronic release of sequestered 5-HT prolongs receptor desensitization. PMID:25281748

  4. Stability of tramadol with three 5-HT3 receptor antagonists in polyolefin bags for patient-controlled delivery systems

    PubMed Central

    Chen, Fu-chao; Zhu, Jun; Li, Bin; Yuan, Fang-jun; Wang, Lin-hai

    2016-01-01

    Background Mixing 5-hydroxytryptamine-3 (5-HT3) receptor antagonists with patient-controlled analgesia (PCA) solutions of tramadol has been shown to decrease the incidence of nausea and vomiting associated with the use of tramadol PCA for postoperative pain. However, such mixtures are not commercially available, and the stability of the drug combinations has not been duly studied. The study aimed to evaluate the stability of tramadol with three 5-HT3 receptor antagonists in 0.9% sodium chloride injection for PCA administration. Materials and methods Test samples were prepared by adding 1,000 mg tramadol hydrochloride, 8 mg ondansetron hydrochloride, and 6 mg granisetron hydrochloride or 5 mg tropisetron hydrochloride to 100 mL of 0.9% sodium chloride injection in polyolefin bags. The samples were prepared in triplicates, stored at either 25°C or 4°C for 14 days, and assessed using the following compatibility parameters: precipitation, cloudiness, discoloration, and pH. Chemical stability was also determined using a validated high-pressure liquid chromatography method. Results All of the mixtures were clear and colorless throughout the initial observation period. No change in the concentration of tramadol hydrochloride occurred with any of the 5-HT3 receptor antagonists during the 14 days. Similarly, little or no loss of the 5-HT3 receptor antagonists occurred over the 14-day period. Conclusion Our results suggest that mixtures of tramadol hydrochloride, ondansetron hydrochloride, granisetron hydrochloride, or tropisetron hydrochloride in 0.9% sodium chloride injection were physically and chemically stable for 14 days when stored in polyolefin bags at both 4°C and 25°C. PMID:27350741

  5. Inhibition of native 5-HT3 receptor-evoked contractions in guinea pig and mouse ileum by antimalarial drugs.

    PubMed

    Kelley, Stephen P; Walsh, Jacqueline; Kelly, Mark C; Muhdar, Simerjyot; Adel-Aziz, Mohammed; Barrett, Iain D; Wildman, Scott S

    2014-09-01

    Quinine, chloroquine and mefloquine are commonly used to treat malaria, however, with associated gastrointestinal (GI) side-effects. These drugs act as antagonists at recombinant 5-HT3 receptors and modulate gut peristalsis. These gastrointestinal side effects may be the result of antagonism at intestinal 5-HT3 receptors. Ileum from male C57BL/6 mice and guinea pigs was mounted longitudinally in organ baths. The concentration-response curves for 5-HT and the selective 5-HT3 agonist 2-Me-5-HT were obtained with 5-HT (pEC50 = 7.57 ± 0.33, 12) more potent (P = 0.004) than 2-Me-5-HT (pEC50 = 5.45 ± 0.58, n = 5) in mouse ileum. There was no difference in potency of 5-HT (pEC50 = 5.42 ± 0.15, n = 8) and 2-Me-5-HT (pIC50 = 5.01 ± 0.55, n = 11) in guinea pig ileum (P > 0.05). Quinine, chloroquine or mefloquine was applied for 10 min and inhibitions prior to submaximal agonist application. In mouse ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50 = 4.9 ± 0.17, n = 7; 4.76 ± 0.14, n = 5; 6.21 ± 0.2, n = 4, correspondingly) with mefloquine most potent (P < 0.05). Quinine, chloroquine and mefloquine antagonised 2-me-5-HT-induced contractions (pIC50 = 6.35 ± 0.11, n = 8; 4.64 ± 0.2, n = 7; 5.11 ± 0.22, n = 6, correspondingly) with quinine most potent (P < 0.05). In guinea-pig ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50 = 5.02 ± 0.15, n = 6; 4.54 ± 0.1, n = 7; 5.32 ± 0.13, n = 5) and 2-me-5-HT-induced contractions (pIC50 = 4.62 ± 0.25, n = 5; 4.56 ± 0.14, n = 6; 5.67 ± 0.12, n = 4) with chloroquine least potent against 5-HT and mefloquine most potent against 2-me-5-HT (P < 0.05). These results support previous studies identifying anti-malarial drugs as antagonists at recombinant 5-HT3 receptors and may also demonstrate the ability of these drugs to influence native 5-HT3 receptor-evoked contractile responses which may account for their associated GI side-effects. PMID:24886883

  6. Synthesis and evaluation of (S)-[(18)F]fesetron in the rat brain as a potential PET imaging agent for serotonin 5-HT3 receptors.

    PubMed

    Pithia, Neema K; Liang, Christopher; Pan, Xiang-Zuo; Pan, Min-Liang; Mukherjee, Jogeshwar

    2016-04-15

    Serotonin 5-HT3 receptors are involved in various brain functions including as an emesis target during cancer chemotherapy. We report here the development of (S)-2,3-dimethoxy-5-(3'-[(18)F]fluoropropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide ([(18)F]fesetron) as a potential PET imaging agent for serotonin 5-HT3 receptors. By radiolabeling((S)-2,3-dimethoxy-5-(3'-tosyloxypropyl)-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide) with fluorine-18, (S)-[(18)F]fesetron was obtained in 5 to 10% decay-corrected yields and with specific activities >74GBq/μmol at the end of radiosynthesis. PET imaging in rats showed low uptake of [(18)F]fesetron in the brain with retention of binding in the striatal and cerebellar regions. Using colliculi as a reference region, ratios were 3.4 for striata and 2.5 for cerebellum. Ex vivo brain PET analysis displayed binding of [(18)F]fesetron in the hippocampus, striatum and cerebellar regions. Cerebellar regions corresponded to area postrema and nucleus tract solitaris known to contain 5-HT3 receptors. Dorsal hippocampus showed the highest uptake with ratio of >17 with respect to colliculi, while area postrema and striata had ratios of >10. Thus, [(18)F]fesetron exhibited a unique binding profile to rat brain regions known to contain significant amounts of serotonin 5-HT3 receptors. However, the very low brain uptake limits its usefulness as a PET radiotracer in this animal model. PMID:26979158

  7. Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling.

    PubMed

    Jarvis, Gavin E; Barbosa, Roseli; Thompson, Andrew J

    2016-03-01

    Citral, eucalyptol, and linalool are widely used as flavorings, fragrances, and cosmetics. Here, we examined their effects on electrophysiological and binding properties of human 5-HT3 receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. Data were analyzed using nonlinear mixed-effects modeling to account for random variance in the peak current response between oocytes. The oils caused an insurmountable inhibition of 5-HT-evoked currents (citral IC50 = 120 µM; eucalyptol = 258 µM; linalool = 141 µM) and did not compete with fluorescently labeled granisetron, suggesting a noncompetitive mechanism of action. Inhibition was not use-dependent but required a 30-second preapplication. Compound washout caused a slow (∼180 seconds) but complete recovery. Coapplication of the oils with bilobalide or diltiazem indicated they did not bind at the same locations as these channel blockers. Homology modeling and ligand docking predicted binding to a transmembrane cavity at the interface of adjacent subunits. Liquid chromatography coupled to mass spectrometry showed that an essential oil extracted from Lippia alba contained 75.9% citral. This inhibited expressed 5-HT3 receptors (IC50 = 45 µg ml(-1)) and smooth muscle contractions in rat trachea (IC50 = 200 µg ml(-1)) and guinea pig ileum (IC50 = 20 µg ml(-1)), providing a possible mechanistic explanation for why this oil has been used to treat gastrointestinal and respiratory ailments. These results demonstrate that citral, eucalyptol, and linalool inhibit 5-HT3 receptors, and their binding to a conserved cavity suggests a valuable target for novel allosteric modulators. PMID:26669427

  8. The Role of Hippocampal 5HT3 Receptors in Harmaline-Induced Memory Deficit

    PubMed Central

    Nasehi, Mohammad

    2015-01-01

    Introduction: The plethora of studies indicated that there is a cross talk relationship between harmaline and serotonergic (5-HT) system on cognitive and non-cognitive behaviors. Thus, the purpose of this study is to assess the effects of hippocampal 5-HT4 receptor on memory acquisition deficit induced by harmaline. Methods: Harmaline was injected peritoneally, while 5-HT4 receptor agonist (RS67333) and antagonist (RS23597-190) were injected intra-hippocampal. A single-trial step-down passive avoidance, open field and tail flick tasks were used for measurement of memory, locomotor activity and pain responses, respectively. Results: The data revealed that pre-training injection of higher dose of harmaline (1 mg/kg), RS67333 (0.5 ng/mouse) and RS23597-190 (0.5 ng/mouse) decreased memory acquisition process in the adult mice. Moreover, concurrent pre-training administration of subthreshold dose of RS67333 (0.005 ng/mouse) or RS23597-190 (0.005 ng/mouse) with subthreshold dose of harmaline (0.5 mg/kg, i.p.) intensify impairment of memory acquisition. All above interventions did not change locomotion and tail flick behaviors. Discussion: The results demonstrated that the synergistic effect between both hippocampal 5-HT4 receptor agonist and antagonist with impairment of memory acquisition induced by harmaline, indicating a modulatory effect for hippocampal 5HT4 receptor on Harmaline induced amnesia. PMID:26904173

  9. Synthesis and biochemical evaluation of tritium-labeled 1-methyl-N-(8-methyl-8-azabicyclo(3. 2. 1)oct-3-yl)-1H-indazole-3-carboxa mide, a useful radioligand for 5HT3 receptors

    SciTech Connect

    Robertson, D.W.; Bloomquist, W.; Cohen, M.L.; Reid, L.R.; Schenck, K.; Wong, D.T. )

    1990-12-01

    The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in (3H)-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of (3H)-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

  10. Comparison of the effects of trimebutine and YM114 (KAE-393), a novel 5-HT3 receptor antagonist, on stress-induced defecation.

    PubMed

    Miyata, K; Ito, H; Yamano, M; Hidaka, K; Kamato, T; Nishida, A; Yuki, H

    1993-12-01

    YM114 (KAE-393), (R)-5-[(2,3-dihydro-1-indolyl)carbonyl]-4,5,6,7- tetrahydro-1H-benzimidazole hydrochloride, is a derivative of YM060, a potent 5-HT3 receptor antagonist. We investigated the effects of YM114 on 5-HT3 receptors, both in vitro and in vivo, and on bowel dysfunction induced by restraint stress, 5-HT and thyrotropin-releasing hormone (TRH), and compared them with the effect of trimebutine. YM114 dose dependently inhibited the reduction in heart rate induced by 5-HT (30 micrograms/kg i.v.) in rats (ED50 = 0.31 micrograms/kg i.v.), and the potency of YM114 was almost the same as that of the racemate. The S-form of YM114 also inhibited 5-HT-induced bradycardia, but 1350 times less potent than the R-form. YM114 and its S-form inhibited [3H]GR65630 binding to N1E-115 cell membranes in a concentration-dependent manner with Ki values of 0.341 and 616 nM, respectively, showing the isomeric activity ratio (R-/S-form) of YM114 to be much greater (1800). YM114 antagonized 5-HT-induced depolarization of the nodose ganglion (EC50 = 1.39 nM). Trimebutine (1 mg/kg i.v.) failed to inhibit 5-HT-induced bradycardia, implying that it does not possess 5-HT3 receptor antagonistic activity. YM114 significantly and dose dependently prevented restraint stress-, 5-HT- and TRH-induced increases in fecal pellet output, and restraint stress- and 5-HT-induced diarrhea in rats and mice (ED50 = 6.9, 72.5, 154.6, 9.7 and 52.4 micrograms/kg p.o., respectively). Trimebutine significantly prevented stress- and 5-HT-induced diarrhea (ED50 = 29.4 and 87.3 mg/kg p.o., respectively), but only partially affected stress-, 5-HT- and TRH-induced increases in fecal pellet output. Thus, YM114 is a potent and stereoselective 5-HT3 receptor antagonist with much greater protective effects against stress-induced defecation than trimebutine. PMID:8112388

  11. [NEPHROPROTECTIVE PROPERTIES OF 5-HT3 RECEPTOR BLOCKER RU-63 IN EXPERIMENTAL ACUTE RENAL FAILURE UNDER HYPERGRAVITY CONDITIONS].

    PubMed

    Zaitseva, E N; Dubishchev, A V; Yakovlev, D S; Anisimova, V A

    2016-01-01

    The effective diuretic dose of 5-HT3 receptor blocker RU-63 (1 mg/kg) was found in experiments on white rats. It is established that the diuretic and saluretic effects of compound RU-63 increase on the background of impact of the gravitational factor. Compound RU-63 (1 mg/kg, subcutaneously) administered daily under hypergravity conditions (3 g in the direction of centrifugal force toward the kidneys) in animals with model ischemic acute renal failure increased excretory function of kidneys, glomerular filtration rate, and creatininuresis (on average by 180%; p < 0.05), and decreased serum creatinine, urinary excretion of protein, lactate dehydrogenase, and g-glutamyl transferase (on average by 49%; p < 0.05) as compared to the untreated control. Under similar conditions, the diuretic hydrochlorothiazide (in a dose of 20 mg/kg, intragastric) produced a more pronounced creatininuretic action than that of RU-63 (by 358%; p < 0.05). PMID:27455574

  12. The role of the AMPA receptor and 5-HT(3) receptor on aggressive behavior and depressive-like symptoms in chronic social isolation-reared mice.

    PubMed

    Shimizu, Koh; Kurosawa, Natsuki; Seki, Kenjiro

    2016-01-01

    Chronic social isolation (SI)-reared mice exhibit aggressive and depressive-like behaviors. However, the pathophysiological changes caused by chronic SI remain unclear. The hypothalamus and amygdala have been suggested to be associated with the stress of SI. In addition to serotonin 3 (5-HT3) receptors, AMPA receptors have also been suggested to be involved in aggressive behavior and depressive-like symptoms in animals. Therefore, we examined whether chronic SI affects AMPA and 5-HT3 receptor expression levels in these regions. A Western blot analysis revealed that after four weeks of SI, mice exhibited up-regulated AMPA receptor subunit (GluR1, GluR2) protein levels in the amygdala and down-regulated hypothalamic 5-HT3 receptor protein levels. The AMPA/kainate receptor antagonist NBQX (10 mg/kg; i.p.) attenuated SI-induced depressive-like symptoms but not aggressive behavior. Intra-amygdalar infusions of the selective AMPA receptor agonist (S)-AMPA (10 μM) induced despair-like behavior, but not sucrose preference or aggressive behavior, in mice not reared in SI (naïve mice). Alternatively, treatment with the 5-HT3 receptor agonist SR57227A (3.0 mg/kg; i.p.) decreased aggression levels. In addition, intra-hypothalamic infusions of the 5-HT3 receptor antagonist ondansetron (3 μM) did not trigger aggressive behavior in naïve mice; however, the administration of ondansetron (0.3 mg/kg; i.p.) increased aggression levels in two-week SI mice, which rarely exhibited the aggressive behavior. Moreover, ondansetron did not affect the depressive-like symptoms of the SI mice. These results suggest that SI-induced up-regulation of GluR1 and GluR2 subunits protein levels in the amygdalar region and down-regulation of 5-HT3 receptor proteins level in the hypothalamic region are associated with the effect of AMPA receptor agonist and 5-HT3 receptor antagonist -induced aggressive behavior and depressive-like symptoms. PMID:26522741

  13. Solid gastric emptying mediated by the serotonin (5-HT)3 receptor in mice is a simple marker to predict emesis.

    PubMed

    Ando, Kentaro; Takagi, Kan

    2011-01-01

    Nausea and emesis are often observed as side effects with many medicines and may lead to poor treatment compliance. In the present study, we aimed to establish simple methods for predicting nausea and/or emesis in mice, which do not vomit, using drugs and chemicals known to evoke nausea and/or emesis. The gastrointestinal transit test, the liquid gastric emptying by phenol red solution (Phenol red method) and the solid gastric emptying by resin beads (Beads method) were used and the effects of antispasmogenics (atropine, 0.1-3 mg/kg i.p.; salmon calcitonin, 1-30 units/kg i.m.), nauseants (copper sulfate, 1-30 mg/kg p.o.; apomorphine, 0.01-0.3 mg/kg s.c.) and chemotherapeutics (cisplatin, 0.3-10 mg/kg i.v.; doxorubicin, 0.3-10 mg/kg i.v.) were evaluated. In addition, the effects of ondansetron, a serotonin (5-HT)(3) receptor antagonist, on the inhibition of solid gastric emptying induced by salmon calcitonin, copper sulfate, cisplatin and doxorubicin were also assessed. Only the solid gastric emptying method could detect changes of gastric emptying by all drugs and chemicals. We also found that the inhibition of solid gastric emptying induced by cisplatin and doxorubicin was dose-dependently antagonized by ondansetron. However, ondansetron failed to antagonize the salmon calcitonin-induced delay, but exerted only very weak effects with copper sulfate. Solid gastric emptying may be more suitable than gastrointestinal intestinal transit or liquid gastric emptying in mice to predict nausea and/or emesis. Our results also suggest that chemotherapeutic-induced delay of solid gastric emptying mediated via 5-HT(3) receptors in mice could also be useful for prediction purposes. PMID:21297338

  14. 5HT3 receptor antagonist (ondansetron) reverses depressive behavior evoked by chronic unpredictable stress in mice: modulation of hypothalamic-pituitary-adrenocortical and brain serotonergic system.

    PubMed

    Gupta, Deepali; Radhakrishnan, Mahesh; Kurhe, Yeshwant

    2014-09-01

    Chronic stress is one of the major causes of depression, associated with behavioral and biochemical impairments. 5HT3 receptor antagonists (such as ondansetron) have shown alleviation of depressive symptomology in preclinical and in few clinical studies. However, their effects in chronic stress-induced depressive behavior and the underlying mechanism(s) are yet to be known. In the present study, the effects of a 5HT3 receptor antagonist, ondansetron were evaluated in chronic unpredictable stress (CUS)-evoked depressive behavior. In addition, the possible mechanism was determined by measuring plasma corticosterone (CORT) as a marker of hypothalamic-pituitary-adrenocortical (HPA)-axis activity and serotonin levels in the discrete brain regions. Mice were subjected to a battery of unpredictable stressors for 28 days. Ondansetron (0.05, 0.1 and 1mg/kg, p.o.) and fluoxetine (10mg/kg, p.o.) were administered during the last 14 days (day 15-28th) of CUS testing paradigm. The results showed that the 4-week CUS produced significant depressive behavior in mice, which included increased despair effects in forced swim test (FST) and reward-related deficits in sucrose preference test. Biochemical assays demonstrated a significant increase in percentage of plasma CORT and decrease in percentage of serotonin levels in the discrete brain regions of CUS mice. Chronic ondansetron treatment, similar to that of positive control fluoxetine, significantly reversed despair effects in FST and reward-related deficits in sucrose preference test. In addition, ondansetron and fluoxetine treatments significantly increased percentage of serotonin levels in the measured brain regions and attenuated HPA-axis hyperactivity, as evidenced by low percentage of plasma CORT levels in CUS mice. These findings indicate the potential role of ondansetron (a 5HT3 receptor antagonist) in reversing CUS-induced depressive behavior, which is possibly mediated by its modulating effects on the HPA-axis and

  15. Different efficacy of specific agonists at 5-HT3 receptor splice variants: the role of the extra six amino acid segment

    PubMed Central

    Niemeyer, M-I; Lummis, S C R

    1998-01-01

    Whole cell voltage clamp electrophysiology and radioligand binding were used to examine the agonist characteristics of the two splice variants of the 5-HT3 receptor which have been cloned from neuronal cell lines. Homo-oligomeric 5-HT3 receptors were examined in HEK 293 cells stably transfected with either long (5-HT3-L) or short (5-HT3-S) receptor subunit DNAs. Functional homo-oligomeric receptors were formed from both subunits, and responses to 5-HT3 receptor agonists (5-hydroxytryptamine (5-HT), 2-methyl 5-HT and m-chlorophenylbiguanide) were qualitatively similar. Maximum currents (Rmax) elicited by the 5-HT3 receptor agonists m-chlorophenylbiguanide (mCPBG) and 2-methyl-5-HT (2-Me-5-HT), as compared to 5-HT, differed in the two splice variants: Rmax mCPBG/Rmax 5-HT values were 0.68±0.04 and 0.91±0.01 in 5-HT3-L and 5-HT3-S receptors, respectively. Comparable values for 2-Me-5-HT were 0.30±0.02 and 0.23±0.02. Radioligand binding data showed no difference in affinity of agonist or antagonist binding sites; thus the six amino acid deletion appears to cause differences in agonist efficacy. The role of the 6 amino acid insertion was further investigated by use of site-directed mutagenesis to create two mutant receptors, one where serine 286 was replaced with alanine, and the second where all 6 amino acids were replaced with alanines. Examination of the mutant receptors when stably expressed in HEK 293 cells revealed agonist properties resembling long and not short 5-HT3 receptors. Thus specific amino acids in this region are not responsible for the observed differences. The data show intracellular structure can have significant effects on ligand-gated ion channel function, and suggest that minor changes in structure may be responsible for differences in function observed when ligand-gated ion channel proteins are modulated intracellularly. PMID:9517385

  16. Length and Amino Acid Sequence of Peptides Substituted for the 5-HT3A Receptor M3M4 Loop May Affect Channel Expression and Desensitization

    PubMed Central

    McKinnon, Nicole K.; Bali, Moez; Akabas, Myles H.

    2012-01-01

    5-HT3A receptors are pentameric neurotransmitter-gated ion channels in the Cys-loop receptor family. Each subunit contains an extracellular domain, four transmembrane segments (M1, M2, M3, M4) and a 115 residue intracellular loop between M3 and M4. In contrast, the M3M4 loop in prokaryotic homologues is <15 residues. To investigate the limits of M3M4 loop length and composition on channel function we replaced the 5-HT3A M3M4 loop with two to seven alanine residues (5-HT3A-An = 2–7). Mutants were expressed in Xenopus laevis oocytes and characterized using two electrode voltage clamp recording. All mutants were functional. The 5-HT EC50's were at most 5-fold greater than wild-type (WT). The desensitization rate differed significantly among the mutants. Desensitization rates for 5-HT3A-A2, 5-HT3A-A4, 5-HT3A-A6, and 5-HT3A-A7 were similar to WT. In contrast, 5-HT3A-A3 and 5-HT3A-A5 had desensitization rates at least an order of magnitude faster than WT. The one Ala loop construct, 5-HT3A-A1, entered a non-functional state from which it did not recover after the first 5-HT application. These results suggest that the large M3M4 loop of eukaryotic Cys-loop channels is not required for receptor assembly or function. However, loop length and amino acid composition can effect channel expression and desensitization. We infer that the cytoplasmic ends of the M3 and M4 segments may undergo conformational changes during channel gating and desensitization and/or the loop may influence the position and mobility of these segments as they undergo gating-induced conformational changes. Altering structure or conformational mobility of the cytoplasmic ends of M3 and M4 may be the basis by which phosphorylation or protein binding to the cytoplasmic loop alters channel function. PMID:22539982

  17. Differential effects of a short-term high-fat diet in an animal model of depression in rats treated with the 5-HT3 receptor antagonist, ondansetron, the 5-HT3 receptor agonist, 2-methyl-5-HT, and the SSRI, fluoxetine.

    PubMed

    Sumaya, Isabel C; Bailey, Dee; Catlett, Susan L

    2016-05-01

    Investigation into the effects of a high-fat diet on depression in the context of 5-HT3 receptor function is important given 5-HT3 antagonism may represent a novel candidate for drug discovery. To more fully understand the relationship between the 5-HT3 receptor system, depression, and high-fat intake, our main interest was to study the short-term effects of a high-fat diet on the 5-HT3 receptor antagonist, ondansetron, and the 5-HT3 receptor agonist, 2-methyl-5-HT, as well as the SSRI, fluoxetine, in an animal model of depression. Male Sprague Dawley rats were fed either a standard diet (11% fat) or a high-fat diet (32.5% fat) for seven days then treated with either fluoxetine (10mg/kg, ip), ondansetron (1mg/kg, ip), 2-methyl-5-HT (3mg/kg, ip), fluoxetine+ondansetron or, 2-methyl-5-HT+ondansetron prior to the Forced Swim Test. In the standard diet group, treatment with the 5HT3 receptor agonist, 2-methyl-5-HT, served to significantly decrease time of immobility as compared to controls thus showing anti-depressive-like effects. Treatment with the 5-HT3 receptor antagonist, ondansetron, served to enhance the anti-depressive like effects of the SSRI, fluoxetine, as treatment with both the SSRI and 5-HT3 receptor antagonist dramatically decreased immobility. Importantly, in the high-fat diet groups, a week of high-fat intake served to: 1) counteract the anti-depressive-like effect of the SSRI, fluoxetine, 2) reverse the anti-depressive-like effect of the 5HT3 receptor agonist, 2-methyl-5-HT and 3) provide protection against the depressive-like effects induced by the Forced Swim Test as rats fed a high-fat diet displayed the lowest amounts of immobility. In the aggregate, these data suggest that both SSRIs and the 5HT3 receptor system are affected by short-term high-fat intake and that a short-term high-fat diet protects against depressive-like effects in an animal model of depression. PMID:26979154

  18. Identification of domains influencing assembly and ion channel properties in α7 nicotinic receptor and 5-HT3 receptor subunit chimaeras

    PubMed Central

    Gee, V J; Kracun, S; Cooper, S T; Gibb, A J; Millar, N S

    2007-01-01

    Background and purpose: Nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are members of the superfamily of neurotransmitter-gated ion channels. Both contain five subunits which assemble to form either homomeric or heteromeric subunit complexes. With the aim of identifying the influence of subunit domains upon receptor assembly and function, a series of chimaeras have been constructed containing regions of the neuronal nAChR α7 subunit and the 5-HT3 receptor 3A subunit. Experimental approach: A series of subunit chimaeras containing α7 and 5-HT3A subunit domains have been constructed and expressed in cultured mammalian cells. Properties of the expressed receptors have been examined by means of radioligand binding, agonist-induced changes in intracellular calcium and patch-clamp electrophysiology. Key results: Subunit domains which influence properties such as rectification, desensitization and conductance have been identified. In addition, the influence of subunit domains upon subunit folding, receptor assembly and cell-surface expression has been identified. Co-expression studies with the nAChR-associated protein RIC-3 revealed that, in contrast to the potentiating effect of RIC-3 on α7 nAChRs, RIC-3 caused reduced levels of cell-surface expression of some α7/5-HT3A chimaeras. Conclusions and implications: Evidence has been obtained which demonstrates that subunit transmembrane domains are critical for efficient subunit folding and assembly. In addition, functional characterization of subunit chimaeras revealed that both extracellular and cytoplasmic domains exert a dramatic and significant influence upon single-channel conductance. These data support a role for regions other than hydrophobic transmembrane domains in determining ion channel properties. PMID:17721553

  19. Modulation of nicotinic ACh-, GABAA- and 5-HT3-receptor functions by external H-7, a protein kinase inhibitor, in rat sensory neurones

    PubMed Central

    Hu, Hong-Zhen; Li, Zhi-Wang

    1997-01-01

    The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GABAA)-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5′-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. External H-7 (1–1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were insensitive to external H-7. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0±4.0 μM to 18.0±5.0 μM). The maximum response to GABA was depressed by 34.0±5.3%. This inhibitory action of H-7 was voltage-independent. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. The results suggest that external H-7 selectively and allosterically modulates the functions of GABAA-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors. PMID:9401786

  20. Evaluation of gene expression changes of serotonin receptors, 5-HT3AR and 5-HT2AR as main stress factors in breast cancer patients.

    PubMed

    Hejazi, Seyed Hesam; Ahangari, Ghasem; Pornour, Majid; Deezagi, Abdolkhaleagh; Aminzadeh, Saeed; Ahmadkhaniha, Hamid Reza; Akbari, Mohamad Esmail

    2014-01-01

    Breast cancer is a serious and potentially lethal multi-factor disease among 40-50 aged women in both developed and developing countries. Also, various studies have pointed to roles of neurotransmitters like serotonin in development of cancers, through action on various types of receptors. This study was conducted to evaluate serotonin receptor (5HT2AR and 5HT3AR) genes expression in peripheral blood mononuclear cells (PBMCs) of breast cancer patients in comparison with the healthy people and in the MCF7 cell line. Peripheral blood samples were obtained from 30 patients and 30 healthy individuals. Total RNA was extracted from PBMCs and MCF-7 cells. and 5HT2AR and 5HT3AR were detected by RT-PCR techniques. Finally, serotonin receptor gene expression variation in breast cancer patients and MCF-7 cells were determined by real time-PCR. This latter indicated significant promotion in expression of 5HT3AR and 5HT2AR in PBMCs in breast cancer patients but expression of 5HT2AR in the MCF-7 cell line was significantly decreased. In conclusion, after performing complimentary tests, determine of gene expression changes in serotonin receptors (5HT2AR and 5HT3AR) may be useful as a new approach in treatment of breast cancer based on use of antagonists. PMID:24969868

  1. Modes and nodes explain the mechanism of action of vortioxetine, a multimodal agent (MMA): blocking 5HT3 receptors enhances release of serotonin, norepinephrine, and acetylcholine.

    PubMed

    Stahl, Stephen M

    2015-10-01

    Vortioxetine is an antidepressant with multiple pharmacologic modes of action at targets where serotonin neurons connect with other neurons. 5HT3 receptor antagonism is one of these actions, and this leads to increased release of norepinephrine (NE), acetylcholine (ACh), and serotonin (5HT) within various brain circuits. PMID:26122791

  2. Role of 5-HT3 receptors in basal and K(+)-evoked dopamine release from rat olfactory tubercle and striatal slices.

    PubMed Central

    Zazpe, A; Artaiz, I; Del Río, J

    1994-01-01

    1. The present study was aimed at examining the role of 5-HT3 receptors in basal and depolarization-evoked dopamine release from rat olfactory tubercle and striatal slices. [3H]-dopamine ([3H]-DA) release was measured in both brain regions and endogenous dopamine release from striatal slices was also studied. 2. The selective 5-HT3 receptor agonist 2-methyl-5-HT (0.5-10 microM) produced a concentration-dependent increase in [3H]-DA efflux evoked by K+ (20 mM) from slices of rat olfactory tubercle. 1-Phenylbiguanide (PBG) and 5-HT also increased K(+)-evoked [3H]-DA efflux. 3. 5-HT (1-100 microM) increased in a concentration-dependent manner basal [3H]-DA release from olfactory tubercle and striatal slices as well as endogenous DA release from striatal slices. The selective 5-HT3 receptor agonists 2-methyl-5-HT and 1-phenylbiguanide were weaker releasing agents. In all cases, the release was Ca2+ independent and tetrodotoxin insensitive. 4. 5-HT3 receptor antagonists such as ondansetron, granisetron and tropisetron (0.2 microM) significantly blocked the enhanced K(+)-evoked [3H]-DA efflux from rat olfactory tubercle slices induced by 2-methyl-5HT. A ten fold higher concentration of the 5-HT2 receptor antagonist ketanserin was ineffective. 5. Much higher concentrations, up to 50 microM, of the same 5-HT3 receptor antagonists did not block the increase in basal [3H]-DA release from striatal or olfactory tubercle slices induced by 5-HT or the release of endogenous DA induced by 5-HT from striatal slices.2+ off PMID:7858893

  3. Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors.

    PubMed

    Pertz, Heinz H; Lehmann, Jochen; Roth-Ehrang, René; Elz, Sigurd

    2011-07-01

    The herbal drug ginger (Zingiber officinale Roscoe) may be effective for treating nausea, vomiting, and gastric hypomotility. In these conditions, cholinergic M (3) receptors and serotonergic 5-HT (3) and 5-HT (4) receptors are involved. The major chemical constituents of ginger are [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol. We studied the interaction of [6]-gingerol, [8]-gingerol, [10]-gingerol (racemates), and [6]-shogaol with guinea pig M (3) receptors, guinea pig 5-HT (3) receptors, and rat 5-HT (4) receptors. In whole segments of guinea pig ileum (bioassay for contractile M (3) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol slightly but significantly depressed the maximal carbachol response at an antagonist concentration of 10 µM. In the guinea pig myenteric plexus preparation (bioassay for contractile 5-HT (3) receptors), 5-HT maximal responses were depressed by [10]-gingerol from 93 ± 3 % to 65 ± 6 % at an antagonist concentration of 3 µM and to 48 ± 3 % at an antagonist concentration of 5 µM following desensitization of 5-HT (4) receptors and blockade of 5-HT (1) and 5-HT (2) receptors. [6]-Shogaol (3 µM) induced depression to 61 ± 3 %. In rat esophageal tunica muscularis mucosae (bioassay for relaxant 5-HT (4) receptors), [6]-gingerol, [8]-gingerol, [10]-gingerol, and [6]-shogaol (2-6.3 µM) showed no agonist effects. The maximal 5-HT response remained unaffected in the presence of the compounds. It is concluded that the efficiency of ginger in reducing nausea and vomiting may be based on a weak inhibitory effect of gingerols and shogaols at M (3) and 5-HT (3) receptors. 5-HT (4) receptors, which play a role in gastroduodenal motility, appear not to be involved in the action of these compounds. PMID:21305447

  4. Combinatorial Consensus Scoring for Ligand-Based Virtual Fragment Screening: A Comparative Case Study for Serotonin 5-HT(3)A, Histamine H(1), and Histamine H(4) Receptors.

    PubMed

    Schultes, Sabine; Kooistra, Albert J; Vischer, Henry F; Nijmeijer, Saskia; Haaksma, Eric E J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2015-05-26

    In the current study we have evaluated the applicability of ligand-based virtual screening (LBVS) methods for the identification of small fragment-like biologically active molecules using different similarity descriptors and different consensus scoring approaches. For this purpose, we have evaluated the performance of 14 chemical similarity descriptors in retrospective virtual screening studies to discriminate fragment-like ligands of three membrane-bound receptors from fragments that are experimentally determined to have no affinity for these proteins (true inactives). We used a complete fragment affinity data set of experimentally determined ligands and inactives for two G protein-coupled receptors (GPCRs), the histamine H1 receptor (H1R) and the histamine H4 receptor (H4R), and one ligand-gated ion channel (LGIC), the serotonin receptor (5-HT3AR), to validate our retrospective virtual screening studies. We have exhaustively tested consensus scoring strategies that combine the results of multiple actives (group fusion) or combine different similarity descriptors (similarity fusion), and for the first time systematically evaluated different combinations of group fusion and similarity fusion approaches. Our studies show that for these three case study protein targets both consensus scoring approaches can increase virtual screening enrichments compared to single chemical similarity search methods. Our cheminformatics analyses recommend to use a combination of both group fusion and similarity fusion for prospective ligand-based virtual fragment screening. PMID:25815783

  5. Synergistic effect between prelimbic 5-HT3 and CB1 receptors on memory consolidation deficit in adult male Sprague-Dawley rats: An isobologram analysis.

    PubMed

    Ahmadi-Mahmoodabadi, N; Nasehi, M; Emam Ghoreishi, M; Zarrindast, M-R

    2016-03-11

    The serotonergic system has often been defined as a neuromodulator system, and is specifically involved in learning and memory via its various receptors. Serotonin is involved in many of the same processes affected by cannabinoids. The present study investigated the influence of bilateral post-training intra-prelimbic (PL) administrations of serotonergic 5-hydroxytryptamine type-3 (5-HT3) receptor agents on arachidonylcyclopropylamide (ACPA) (cannabinoid CB1 receptor agonist)-induced amnesia, using the step-through inhibitory avoidance (IA) task to assess memory in adult male Sprague-Dawley rats. The results indicated that sole intra-PL microinjection of ACPA (0.1 and 0.5μg/rat) and 5-HT3 serotonin receptor agonist (m-Chlorophenylbiguanide hydrochloride, m-CPBG; 0.001, 0.01 and 0.1μg/rat) impaired, whereas Y-25130 (a selective 5-HT3 serotonin receptor antagonist; 0.001 and 0.01 and 0.1μg/rat) did not alter IA memory consolidation, by itself. Moreover, intra-PL administration of subthreshold dose of m-CPBG (0.0005μg/rat) potentiated, while Y-25130 (0. 1μg/rat) restored ACPA-induced memory consolidation deficit. The isobologram analysis showed that there is a synergistic effect between ACPA and m-CPBG on memory consolidation deficit. These findings suggest that 5-HT3 receptor mechanism(s), at least partly, play(s) a role in modulating the effect of ACPA on memory consolidation in the PL area. PMID:26701293

  6. Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes

    PubMed Central

    Mair, Ian D; Lambert, Jeremy J; Yang, Jay; Dempster, John; Peters, John A

    1998-01-01

    The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM–10 μM) evoked a transient, concentration-dependent (EC50=1.1±0.1 μM), inward current. The response reversed in sign at a holding potential of −2.1±1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50=4.1±0.2 μM), 1-phenylbiguanide (EC50=3.0±0.1 μM), 3-chlorophenylbiguanide (EC50=140± 10 nM), 3,5-dichlorophenylbiguanide (EC50=14.5±0.4 nM) and 2,5-dichlorophenylbiguanide (EC50= 10.2±0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 μM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231±22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9± 0.01 nM) and cocaine (IC50=2.1±0.2 μM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues. PMID:9756382

  7. Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

    PubMed

    Youn, Ui Joung; Lee, Jun-Ho; Lee, Yoo Jin; Nam, Joo Won; Bae, Hyunsu; Seo, Eun-Kyoung

    2010-09-01

    Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes. PMID:20860031

  8. Role of peripheral and spinal 5-HT(3) receptors in development and maintenance of formalin-induced long-term secondary allodynia and hyperalgesia.

    PubMed

    Bravo-Hernández, Mariana; Cervantes-Durán, Claudia; Pineda-Farias, Jorge Baruch; Barragán-Iglesias, Paulino; López-Sánchez, Pedro; Granados-Soto, Vinicio

    2012-04-01

    The role of peripheral and spinal 5-HT(3) receptors in formalin-induced secondary allodynia and hyperalgesia in rats was assessed. Formalin produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-term secondary mechanical allodynia and hyperalgesia in both paws. In experiments where the test drug was anticipated to augment or antagonize the response, 0.5 or 1% formalin, respectively, was used for injection. Peripheral ipsilateral, but not contralateral, pre-treatment (-10 min) with serotonin (5-HT, 10-100 nmol/paw) and the selective 5-HT(3) receptor agonist 1-(m-chlorophenyl)-biguanide (m-CPBG, 10-300 nmol/paw) increased 0.5% formalin-induced secondary allodynia and hyperalgesia in both paws. Moreover, spinal pre-treatment with m-CPBG (10-300 nmol/rat) increased 0.5% formalin-induced secondary hyperalgesia but not allodynia in both paws. Accordingly, peripheral ipsilateral (30-300 nmol/paw), but not contralateral (300 nmol/paw), and spinal (10-100 nmol) pre-treatment with the selective 5-HT(3) receptor antagonist ondansetron prevented 1% formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. The peripheral pronociceptive effects of 5-HT (100 nmol/paw) and m-CPBG (300 nmol/paw) as well as the spinal effect of m-CPBG (300 nmol/rat) were completely prevented by the peripheral (10 nmol/paw) and spinal (1 nmol/rat) injection, respectively, of ondansetron. At these doses, ondansetron did not modify per se formalin-induced nociceptive behaviors. Spinal (30-300 nmol/rat), but not peripheral (300 nmol/paw), post-treatment (on day 6) with ondansetron reversed established formalin-induced secondary mechanical allodynia and hyperalgesia in both paws. Results suggest that a barrage of afferent input induced by 5-HT at peripheral 5-HT(3) receptors participates in the development of formalin-induced long-term secondary allodynia and hyperalgesia in the rat. In addition, our data suggest that spinal 5-HT(3) receptors play an

  9. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-06-15

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11439930

  10. Conversion of the ion selectivity of the 5-HT(3a) receptor from cationic to anionic reveals a conserved feature of the ligand-gated ion channel superfamily.

    PubMed

    Gunthorpe, M J; Lummis, S C

    2001-04-01

    The 5-hydroxytryptamine(3) (5-HT(3)) receptor is a member of a superfamily of ligand-gated ion channels, which includes nicotinic acetylcholine, gamma-aminobutyric acid, and glycine receptors. The receptors are either cation or anion selective, leading to their distinctive involvement in either excitatory or inhibitory neurotransmission. Using a combination of site-directed mutagenesis and electrophysiological characterization of homomeric 5-HT(3A) receptors expressed in HEK293 cells, we have identified a set of mutations that convert the ion selectivity of the 5-HT(3A) receptor from cationic to anionic; these were substitution of V13'T in M2 together with neutralization of glutamate residues (E-1'A) and the adjacent insertion of a proline residue (P-1') in the M1-M2 loop. Mutant receptors showed significant chloride permeability (P(Cl)/P(Na) = 12.3, P(Na)/P(Cl) = 0.08), whereas WT receptors are predominantly permeable to sodium (P(Na)/P(Cl) > 20, P(Cl)/P(Na) < 0.05). Since the equivalent mutations have previously been shown to convert alpha7 nicotinic acetylcholine receptors from cationic to anionic (Galzi J.-L., Devillers-Thiery, A, Hussy, N., Bertrand, S. Changeux, J. P., and Bertrand, D. (1992) Nature 359, 500-505) and, recently, the converse mutations have allowed the construction of a cation selective glycine receptor (Keramidas, A., Moorhouse, A. J., French, C. R., Schofield, P. R., and Barry, P. H. (2000) Biophys. J. 78, 247-259), it appears that the determinants of ion selectivity represent a conserved feature of the ligand-gated ion channel superfamily. PMID:11139582

  11. Serotonin homeostasis and serotonin receptors as actors of cortical construction: special attention to the 5-HT3A and 5-HT6 receptor subtypes

    PubMed Central

    Vitalis, Tania; Ansorge, Mark S.; Dayer, Alexandre G.

    2013-01-01

    Cortical circuits control higher-order cognitive processes and their function is highly dependent on their structure that emerges during development. The construction of cortical circuits involves the coordinated interplay between different types of cellular processes such as proliferation, migration, and differentiation of neural and glial cell subtypes. Among the multiple factors that regulate the assembly of cortical circuits, 5-HT is an important developmental signal that impacts on a broad diversity of cellular processes. 5-HT is detected at the onset of embryonic telencephalic formation and a variety of serotonergic receptors are dynamically expressed in the embryonic developing cortex in a region and cell-type specific manner. Among these receptors, the ionotropic 5-HT3A receptor and the metabotropic 5-HT6 receptor have recently been identified as novel serotonergic targets regulating different aspects of cortical construction including neuronal migration and dendritic differentiation. In this review, we focus on the developmental impact of serotonergic systems on the construction of cortical circuits and discuss their potential role in programming risk for human psychiatric disorders. PMID:23801939

  12. Mapping Spatial Relationships between Residues in the Ligand-Binding Domain of the 5-Ht3 Receptor Using a Molecular Ruler

    PubMed Central

    Nyce, Heather L.; Stober, Spencer T.; Abrams, Cameron F.; White, Michael M.

    2010-01-01

    Abstract The serotonin 5-HT3 receptor (5-HT3R) is a member of the Cys-loop ligand-gated ion channel family. We used a combination of site-directed mutagenesis, homology modeling, and ligand-docking simulations to analyze antagonist-receptor interactions. Mutation of E236, which is near loop C of the binding site, to aspartate prevents expression of the receptor on the cell surface, and no specific ligand binding can be detected. On the other hand, mutation to glutamine, asparagine, or alanine produces receptors that are expressed on the cell surface, but decreases receptor affinity for the competitive antagonist d-tubocurarine (dTC) 5-35-fold. The results of a double-mutant cycle analysis employing a panel of dTC analogs to identify specific points of interactions between the dTC analogs and E236 are consistent with E236 making a direct physical interaction with the 12 –OH of dTC. dTC is a rigid molecule of known three-dimensional structure. Together with previous studies linking other regions of dTC to specific residues in the binding site, these data allow us to define the relative spatial arrangement of three different residues in the ligand-binding site: R92 (loop D), N128 (loop A), and E236 (near loop C). Molecular modeling employing these distance constraints followed by molecular-dynamics simulations produced a dTC/receptor complex consistent with the experimental data. The use of the rigid ligands as molecular rulers in conjunction with double-mutant cycle analysis provides a means of mapping the relative positions of various residues in the ligand-binding site of any ligand-receptor complex, and thus is a useful tool for delineating the architecture of the binding site. PMID:20441748

  13. Vortioxetine dose-dependently reverses 5-HT depletion-induced deficits in spatial working and object recognition memory: a potential role for 5-HT1A receptor agonism and 5-HT3 receptor antagonism.

    PubMed

    du Jardin, Kristian Gaarn; Jensen, Jesper Bornø; Sanchez, Connie; Pehrson, Alan L

    2014-01-01

    We previously reported that the investigational multimodal antidepressant, vortioxetine, reversed 5-HT depletion-induced memory deficits while escitalopram and duloxetine did not. The present report studied the effects of vortioxetine and the potential impact of its 5-HT1A receptor agonist and 5-HT3 receptor antagonist properties on 5-HT depletion-induced memory deficits. Recognition and spatial working memory were assessed in the object recognition (OR) and Y-maze spontaneous alternation (SA) tests, respectively. 5-HT depletion was induced in female Long-Evans rats using 4-cholro-DL-phenylalanine methyl ester HCl (PCPA) and receptor occupancies were determined by ex vivo autoradiography. Rats were acutely dosed with vortioxetine, ondansetron (5-HT3 receptor antagonist) or flesinoxan (5-HT1A receptor agonist). The effects of chronic vortioxetine administration on 5-HT depletion-induced memory deficits were also assessed. 5-HT depletion reliably impaired memory performance in both the tests. Vortioxetine reversed PCPA-induced memory deficits dose-dependently with a minimal effective dose (MED) ≤0.1mg/kg (∼80% 5-HT3 receptor occupancy; OR) and ≤3.0mg/kg (5-HT1A, 5-HT1B, 5-HT3 receptor occupancy: ∼15%, 60%, 95%) in SA. Ondansetron exhibited a MED ≤3.0μg/kg (∼25% 5-HT3 receptor occupancy; OR), but was inactive in the SA test. Flesinoxan had a MED ≤1.0mg/kg (∼25% 5-HT1A receptor occupancy; SA); only 1.0mg/kg ameliorated deficits in the NOR. Chronic p.o. vortioxetine administration significantly improved memory performance in OR and occupied 95%, 66%, and 9.5% of 5-HT3, 5-HT1B, and 5-HT1A receptors, respectively. Vortioxetine's effects on SA performance may involve 5-HT1A receptor agonism, but not 5-HT3 receptor antagonism, whereas the effects on OR performance may involve 5-HT3 receptor antagonism and 5-HT1A receptor agonism. PMID:23916504

  14. Critical role of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex.

    PubMed

    Dickson, Eamonn J; Heredia, Dante J; Smith, Terence K

    2010-07-01

    The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7

  15. X-ray analysis of the effect of the 5-HT3 receptor antagonist granisetron on gastrointestinal motility in rats repeatedly treated with the antitumoral drug cisplatin.

    PubMed

    Vera, Gema; López-Pérez, Ana Esther; Martínez-Villaluenga, María; Cabezos, Pablo Antonio; Abalo, Raquel

    2014-08-01

    Cancer chemotherapy is associated with the development of numerous adverse effects, including nausea, emesis and other alterations in gastrointestinal (GI) motility. The administration of 5-HT3 receptor antagonists has provided a clinical advance in the treatment of chemotherapy-induced vomiting but these drugs lose efficacy throughout chronic treatment. The effects of these drugs in experimental animals under chronic administration are not well known. Our aim was to study, using radiographic methods, the effect of the 5-HT3 receptor antagonist granisetron on GI dysmotility induced in the rat by repeated cisplatin administration. First, invasive methods were used to select a dose of granisetron capable of reducing increased stomach weight due to acute cisplatin administration (6 mg/kg, ip). Second, rats received two intraperitoneal (ip) injections once a week for 4 weeks: granisetron (1 mg/kg, ip) or saline and, thirty min later, saline or cisplatin (2 mg/kg, ip). Body weight gain was measured throughout treatment. Radiological techniques were used to determine the acute (after first dose) and chronic (after last dose) effects of cisplatin and/or granisetron on GI motility. Repeated cisplatin-induced weight loss which granisetron did not prevent. Gastric emptying was delayed after the first cisplatin administration. Granisetron completely prevented this effect. After weekly administration, cisplatin-induced gastric dysmotility was enhanced and granisetron was not capable of completely preventing this effect. Granisetron prevents gastric emptying alterations, but its efficacy decreases throughout antineoplastic treatment. This might be due to the enhanced effect of cisplatin. PMID:24798399

  16. Effects of serotonin 5-HT3 receptor antagonists on stress-induced colonic hyperalgesia and diarrhoea in rats: a comparative study with opioid receptor agonists, a muscarinic receptor antagonist and a synthetic polymer.

    PubMed

    Hirata, T; Keto, Y; Nakata, M; Takeuchi, A; Funatsu, T; Akuzawa, S; Sasamata, M; Miyata, K

    2008-05-01

    In this study, we examined the effects of serotonin (5-HT)3 receptor antagonists (5-HT3RAs) including ramosetron, alosetron, and cilansetron on colonic nociceptive threshold in rats. Furthermore, we established a restraint stress-induced colonic hyperalgesia model in rats, and compared the inhibitory effects of 5-HT3RAs on restraint stress-induced colonic hyperalgesia and diarrhoea with those of loperamide, trimebutine, tiquizium and polycarbophil. The colonic nociceptive threshold was measured as the balloon pressure at the time the rat showed a nociceptive response during colonic distension by an intrarectally inserted balloon. Oral administration of ramosetron (3-30 microg kg(-1)), alosetron (30-300 microg kg(-1)), or cilansetron (30-300 microg kg(-1)) increased the colonic nociceptive threshold in a dose-dependent manner in non-stressed rats. Restraint stress for 1 h significantly decreased the colonic nociceptive threshold, but ramosetron (0.3-3 microg kg(-1)), alosetron (3-30 microg kg(-1)), cilansetron (3-30 microg kg(-1)) and trimebutine (100-1000 mg kg(-1)) significantly inhibited the decrease in the threshold. Loperamide (3-30 mg kg(-1)), tiquizium (100-1000 mg kg(-1)) and polycarbophil (1000 mg kg(-1)) did not affect the restraint stress-induced decrease in the colonic nociceptive threshold. All drugs tested in this study showed dose-dependent inhibition of restraint stress-induced diarrhoea in rats. These results indicate that, unlike existing antidiarrhoeal and spasmolytic agents, 5-HT3RAs have inhibitory effects on colonic nociception, and prevented restraint stress-induced both diarrhoea and hyperalgesia at almost the same doses in rats. This suggests that the 5-HT3RAs may be useful in ameliorating both colonic hyperalgesia and diarrhoea in patients with irritable bowel syndrome. PMID:18221252

  17. A retrospective study of R-CHOP/CHOP therapy-induced nausea and vomiting in non-Hodgkin's lymphoma patients: a comparison of intravenous and oral 5-HT3 receptor antagonists.

    PubMed

    Takahashi, Tsutomu; Kumanomidou, Satoshi; Takami, Saki; Okada, Takahiro; Adachi, Koji; Jo, Yumi; Ikejiri, Fumiyoshi; Onishi, Chie; Kawakami, Koshi; Miyake, Takaaki; Inoue, Masaya; Moriyama, Ichiro; Suzuki, Ritsuro; Suzumiya, Junji

    2016-09-01

    Chemotherapy-induced nausea and vomiting (CINV) is a serious problem for cancer patients receiving chemotherapy. The CHOP regimen is the standard treatment for non-Hodgkin's lymphoma (NHL) and is categorized as highly or moderately emetogenic in the CINV guidelines. The efficacy of oral 5-HT3 receptor antagonists is equivalent to that of the intravenous form in patients with solid tumors, but there is no clear comparative data for the use of these agents NHL patients receiving CHOP. We analyzed retrospective CINV data from medical records of 72 NHL patients who received CHOP or rituximab-combined CHOP therapy (R-CHOP). All patients received 5-HT3 receptor antagonists alone for prevention of CINV; 39 of the patients received an intravenous form (mostly granisetron) and 33 an oral form (all ramosetron). Complete response (CR: defined as no vomiting and no rescue therapy) was observed in 58 of 72 patients (80.6 %) overall (0-120 h post-CHOP). The CR rate was not statistically different in patients treated with oral or intravenous 5-HT3 receptor antagonists (82.1 vs 78.8 %, P = 0.77). These findings suggest that oral 5-HT3 receptor antagonists represent a good alternative to intravenous forms in NHL receiving CHOP/R-CHOP chemotherapy. Further studies are needed to identify the optimal anti-emetic supportive therapy for NHL. PMID:27312042

  18. Ondansetron reverses anti-hypersensitivity from clonidine in rats following peripheral nerve injury: Role of γ-amino butyric acid in α2-adrenoceptor and 5-HT3 serotonin receptor analgesia

    PubMed Central

    Hayashida, Ken-ichiro; Kimura, Masafumi; Yoshizumi, Masaru; Hobo, Shotaro; Obata, Hideaki; Eisenach, James C.

    2012-01-01

    Introduction Monoaminergic pathways, impinging an α2-adrenoceptors and 5-HT3 serotonin receptors, modulate nociceptive transmission, but their mechanisms and interactions after neuropathic injury are unknown. Here we examine these interactions in rodents after nerve injury. Methods Male Sprague-Dawley rats following L5-L6 spinal nerve ligation (SNL) were used for either behavioral testing, in vivo microdialysis for γ-amino butyric acid (GABA) and acetylcholine release, or synaptosome preparation for GABA release. Results Intrathecal administration of the α2-adrenoceptor agonist (clonidine) and 5-HT3 receptor agonist (chlorophenylbiguanide) reduced hypersensitivity in SNL rats via GABA receptor-mediated mechanisms. Clonidine increased GABA and acetylcholine release in vivo in the spinal cord of SNL rats but not in normal rats. Clonidine-induced spinal GABA release in SNL rats was blocked by α2-adrenergic and nicotinic cholinergic antagonists. The 5-HT3 receptor antagonist ondansetron decreased and chlorophenylbiguanide increased spinal GABA release in both normal and SNL rats. In synaptosomes from the spinal dorsal horn of SNL rats, pre-synaptic GABA release was increased by nicotinic agonists and decreased by muscarinic and α2-adrenergic agonists. Spinally administered ondansetron significantly reduced clonidine-induced anti-hypersensitivity and spinal GABA release in SNL rats. Conclusion These results suggest that spinal GABA contributes to anti-hypersensitivity from intrathecal α2-adrenergic and 5-HT3 receptor agonists in the neuropathic pain state, that cholinergic neuroplasticity after nerve injury is critical for α2-adrenoceptor-mediated GABA release, and that blockade of spinal 5-HT3 receptors reduces α2-adrenoceptor-mediated anti-hypersensitivity via reducing total GABA release. PMID:22722575

  19. Expression of 5-HT3 receptors and TTX resistant sodium channels (NaV1.8) on muscle nerve fibers in pain-free humans and patients with chronic myofascial temporomandibular disorders

    PubMed Central

    2014-01-01

    Background Previous studies have shown that 5-HT3-antagonists reduce muscle pain, but there are no studies that have investigated the expression of 5-HT3-receptors in human muscles. Also, tetrodotoxin resistant voltage gated sodium-channels (NaV) are involved in peripheral sensitization and found in trigeminal ganglion neurons innervating the rat masseter muscle. This study aimed to investigate the frequency of nerve fibers that express 5-HT3A-receptors alone and in combination with NaV1.8 sodium-channels in human muscles and to compare it between healthy pain-free men and women, the pain-free masseter and tibialis anterior muscles, and patients with myofascial temporomandibular disorders (TMD) and pain-free controls. Methods Three microbiopsies were obtained from the most bulky part of the tibialis and masseter muscles of seven and six healthy men and seven and six age-matched healthy women, respectively, while traditional open biopsies were obtained from the most painful spot of the masseter of five female patients and from a similar region of the masseter muscle of five healthy, age-matched women. The biopsies were processed by routine immunohistochemical methods. The biopsy sections were incubated with monoclonal antibodies against the specific axonal marker PGP 9.5, and polyclonal antibodies against the 5-HT3A-receptors and NaV1.8 sodium-channels. Results A similar percentage of nerve fibers in the healthy masseter (85.2%) and tibialis (88.7%) muscles expressed 5-HT3A-receptors. The expression of NaV1.8 by 5-HT3A positive nerve fibers associated with connective tissue was significantly higher than nerve fibers associated with myocytes (P < .001). In the patients, significantly more fibers per section were found with an average of 3.8 ± 3 fibers per section in the masseter muscle compared to 2.7 ± 0.2 in the healthy controls (P = .024). Further, the frequency of nerve fibers that co-expressed NaV1.8 and 5-HT3A receptors was significantly

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

    PubMed

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

    2015-07-23

    Previous electrophysiological studies demonstrated a limited role of 5-hydroxytryptamine 3 receptor (5-HT3R), but facilitatory role of 5-HT1AR and 5-HT1BR in spinal nociceptive processing of carrageenan-induced inflammatory pain. The release of spinal 5-HT was shown to peak in early-phase and return to baseline in late-phase of carrageenan inflammation. We examined the role of the descending serotonergic projections involving 5-HT1AR, 5-HT1BR, and 5-HT3R in mechanical allodynia of early- (first 4h) and late-phase (24h after) carrageenan-induced inflammation. Intrathecal administration of 5-HT produced a significant anti-allodynic effect in late-phase, but not in early-phase. Similarly, intrathecal 5-HT1AR agonist (8-OH-DPAT) attenuated the intensity of late-phase allodynia in a dose dependent fashion which was antagonized by 5-HT1AR antagonist (WAY-100635), but produced no effect on the early-phase allodynia. However, other agonists or antagonists of 5-HT1BR (CP-93129, SB-224289) and 5-HT3R (m-CPBG, ondansetron) did not produce any anti- or pro-allodynic effect in both early- and late- phase allodynia. These results suggest that spinal 5-HT1A, but not 5-HT1B or 5-HT3 receptors mediate descending serotonergic inhibition on nociceptive processing of late-phase mechanical allodynia in carrageenan-induced inflammation. PMID:26037417

  1. Analysis of free ACh and 5-HT in milk from four different species and their bioactivity on 5-HT(3) and nACh receptors.

    PubMed

    Gallegos-Perez, Jose-Luis; Limon, Agenor; Reyes-Ruiz, Jorge M; Alshanqeeti, Ali S; Aljohi, Mohammad A; Miledi, Ricardo

    2014-07-25

    Milk is one of the most beneficial aliments and is highly recommended in normal conditions; however, in certain disorders, like irritable bowel syndrome, cow milk and dairy products worsen the gastric symptoms and their use is not recommended. Among the most recognized milk-induced gatrointestinal symptoms are abdominal pain, nausea and vomiting, which are processes controlled by cholinergic and serotonergic transmission. Whether the presence of bioavailable ACh and 5-HT in milk may contribute to normal peristalsis, or to the developing of these symptoms, is not known. In this work we attempt to determine whether the content of free ACh and 5-HT is of physiological significance in milk from four different species: cow (bovine), goat, camel and human. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was used to identify and quantify free ACh and 5-HT in milk, and activation of the serotonergic and cholinergic ionotropic receptors was investigated using electrophysiological experiments. Our principal hypothesis was that milk from these four species had sufficient free ACh and 5-HT to activate their correspondent receptors expressed in a heterologous system. Our results showed a more complex picture, in which free ACh and 5-HT and their ability to activate cholinergic and serotonergic receptors are not correlated. This work is a first step to elucidate whether 5-HT and ACh, at the concentrations present in the milk, can be associated to a direct function in the GI. PMID:24820623

  2. Sleep Deprivation-Induced Blood-Brain Barrier Breakdown and Brain Dysfunction are Exacerbated by Size-Related Exposure to Ag and Cu Nanoparticles. Neuroprotective Effects of a 5-HT3 Receptor Antagonist Ondansetron.

    PubMed

    Sharma, Aruna; Muresanu, Dafin F; Lafuente, José V; Patnaik, Ranjana; Tian, Z Ryan; Buzoianu, Anca D; Sharma, Hari S

    2015-10-01

    Military personnel are often subjected to sleep deprivation (SD) during combat operations. Since SD is a severe stress and alters neurochemical metabolism in the brain, a possibility exists that acute or long-term SD will influence blood-brain barrier (BBB) function and brain pathology. This hypothesis was examined in young adult rats (age 12 to 14 weeks) using an inverted flowerpot model. Rats were placed over an inverted flowerpot platform (6.5 cm diameter) in a water pool where the water levels are just 3 cm below the surface. In this model, animals can go to sleep for brief periods but cannot achieve deep sleep as they would fall into water and thus experience sleep interruption. These animals showed leakage of Evans blue in the cerebellum, hippocampus, caudate nucleus, parietal, temporal, occipital, cingulate cerebral cortices, and brain stem. The ventricular walls of the lateral and fourth ventricles were also stained blue, indicating disruption of the BBB and the blood-cerebrospinal fluid barrier (BCSFB). Breakdown of the BBB or the BCSFB fluid barrier was progressive in nature from 12 to 48 h but no apparent differences in BBB leakage were seen between 48 and 72 h of SD. Interestingly, rats treated with metal nanoparticles, e.g., Cu or Ag, showed profound exacerbation of BBB disruption by 1.5- to 4-fold, depending on the duration of SD. Measurement of plasma and brain serotonin showed a close correlation between BBB disruption and the amine level. Repeated treatment with the serotonin 5-HT3 receptor antagonist ondansetron (1 mg/kg, s.c.) 4 and 8 h after SD markedly reduced BBB disruption and brain pathology after 12 to 24 h SD but not following 48 or 72 h after SD. However, TiO2-nanowired ondansetron (1 mg/kg, s.c) in an identical manner induced neuroprotection in rats following 48 or 72 h SD. However, plasma and serotonin levels were not affected by ondansetron treatment. Taken together, our observations are the first to show that (i) SD could induce BBB

  3. Inhibitory effects of ramosetron, a potent and selective 5-HT3-receptor antagonist, on conditioned fear stress-induced abnormal defecation and normal defecation in rats: comparative studies with antidiarrheal and spasmolytic agents.

    PubMed

    Hirata, Takuya; Funatsu, Toshiyuki; Keto, Yoshihiro; Akuzawa, Shinobu; Sasamata, Masao; Miyata, Keiji

    2008-02-01

    We examined the effect of ramosetron, a potent serotonin (5-HT)(3)-receptor antagonist for irritable bowel syndrome with diarrhea, on conditioned fear stress (CFS)-induced defecation and normal (non-stressed) defecation in rats and compared ramosetron with the antidiarrheal agent loperamide and the spasmolytic agents trimebutine and tiquizium. Ramosetron, loperamide, trimebutine, and tiquizium significantly inhibited CFS-induced defecation in a dose-dependent manner with ED(50) (95% confidence limit) values of 0.019 (0.01 - 0.028), 9.4 (4.0 - 22), 850 (520 - 2,400), and 300 (190 - 450) mg/kg, respectively. A significant effect of ramosetron on CFS-induced defecation appeared at 10 min after dosing and was sustained for 8 h. In contrast, loperamide, trimebutine, and tiquizium significantly inhibited CFS-induced defecation between 1 - 8, 1 - 4, and 1 - 8 h after administration, respectively. High doses of ramosetron did not affect normal defecation, whereas loperamide, trimebutine, and tiquizium significantly inhibited this process. In conclusion, ramosetron has potent, rapid-onset, and long-lasting inhibitory effects on CFS-induced defecation in rats, but does not influence normal defecation. The present findings indicate that ramosetron will be a useful therapeutic agent for irritable bowel syndrome with diarrhea, showing greater efficacy and safety than other antidiarrheal and spasmolytic agents. PMID:18296863

  4. Effect of the selective 5-HT3 receptor antagonists ICS 205-930 and MDL 72222 on 5-HTP-induced head shaking and behavioral symptoms induced by 5-methoxy-N,N,dimethyltryptamine in rats: comparison with some other 5-HT receptor antagonists.

    PubMed

    Shearman, G T; Tolcsvai, L

    1987-01-01

    The effect of the selective 5-HT3 receptor antagonists ICS 205-930 and MDL 72222 on head shaking behavior induced by L-5-HTP and behavioral symptoms induced with 5-methoxy-N,N,-dimethyltryptamine (5-MeODMT) in rats was evaluated. Both drugs dose-dependently reduced L-5-HTP-induced head shaking but were at least 600 times less potent than pirenperone and ketanserin and at least 50 times less potent than methysergide. ICS 205-930 and MDL 72222 were more than 1000 times less potent than pirenperone or methysergide and 100 times less potent than ketanserin in blocking 5-MeODMT-induced forepaw treading and tremor. Since it appears that head shakes induced by L-5-HTP are mediated by 5-HT2 receptors, these data suggest that ICS 205-930 and MDL 72222 do not significantly interact with 5-HT2 receptors in the brain. Furthermore, the data suggest that ICS 205-930 and MDL 72222 lack appreciable antagonistic activity at the 5-HT receptor(s) mediating those behavioral effects induced by 5-MeODMT. PMID:3114804

  5. Activation of human 5-hydroxytryptamine type 3 receptors via an allosteric transmembrane site.

    PubMed

    Lansdell, Stuart J; Sathyaprakash, Chaitra; Doward, Anne; Millar, Neil S

    2015-01-01

    In common with other members of the Cys-loop family of pentameric ligand-gated ion channels, 5-hydroxytryptamine type 3 receptors (5-HT3Rs) are activated by the binding of a neurotransmitter to an extracellular orthosteric site, located at the interface of two adjacent receptor subunits. In addition, a variety of compounds have been identified that modulate agonist-evoked responses of 5-HT3Rs, and other Cys-loop receptors, by binding to distinct allosteric sites. In this study, we examined the pharmacological effects of a group of monoterpene compounds on recombinant 5-HT3Rs expressed in Xenopus oocytes. Two phenolic monoterpenes (carvacrol and thymol) display allosteric agonist activity on human homomeric 5-HT3ARs (64 ± 7% and 80 ± 4% of the maximum response evoked by the endogenous orthosteric agonist 5-HT, respectively). In addition, at lower concentrations, where agonist effects are less apparent, carvacrol and thymol act as potentiators of responses evoked by submaximal concentrations of 5-HT. By contrast, carvacrol and thymol have no agonist or potentiating activity on the closely related mouse 5-HT3ARs. Using subunit chimeras containing regions of the human and mouse 5-HT3A subunits, and by use of site-directed mutagenesis, we have identified transmembrane amino acids that either abolish the agonist activity of carvacrol and thymol on human 5-HT3ARs or are able to confer this property on mouse 5-HT3ARs. By contrast, these mutations have no significant effect on orthosteric activation of 5-HT3ARs by 5-HT. We conclude that 5-HT3ARs can be activated by the binding of ligands to an allosteric transmembrane site, a conclusion that is supported by computer docking studies. PMID:25338672

  6. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. PMID:26456648

  7. Block of the delayed rectifier current (IK) by the 5-HT3 antagonists ondansetron and granisetron in feline ventricular myocytes.

    PubMed Central

    de Lorenzi, F G; Bridal, T R; Spinelli, W

    1994-01-01

    1. We investigated the effects of two 5-HT3 antagonists, ondansetron and granisetron, on the action potential duration (APD) and the delayed rectifier current (IK) of feline isolated ventricular myocytes. Whole-cell current and action potential recordings were performed at 37 degrees C with the patch clamp technique. 2. Ondansetron and granisetron blocked IK with a KD of 1.7 +/- 1.0 and 4.3 +/- 1.7 microM, respectively. At a higher concentration (30 microM), both drugs blocked the inward rectifier (IKl). 3. The block of IK was dependent on channel activation. Both drugs slowed the decay of IK tail currents and produced a crossover with the pre-drug current trace. These results are consistent with block and unblock from the open state of the channel. 4. Granisetron showed an intrinsic voltage-dependence as the block increased with depolarization. The equivalent voltage-dependency of block (delta) was 0.10 +/- 0.04, suggesting that granisetron blocks from the intracellular side at a binding site located 10% across the transmembrane electrical field. 5. Ondansetron (1 microM) and granisetron (3 microM) prolonged APD by about 30% at 0.5 Hz. The prolongation of APD by ondansetron was abolished at faster frequencies (3 Hz) showing reverse rate dependence. 6. In conclusion, the 5-HT3 antagonists, ondansetron and granisetron, are open state blockers of the ventricular delayed rectifier and show a clear class III action. PMID:7834204

  8. Patient perceptions of the side-effects of chemotherapy: the influence of 5HT3 antagonists.

    PubMed Central

    de Boer-Dennert, M.; de Wit, R.; Schmitz, P. I.; Djontono, J.; v Beurden, V.; Stoter, G.; Verweij, J.

    1997-01-01

    In 1983, Coates conducted a survey that ranked the side-effects perceived by patients receiving chemotherapy in the order of their severity. Vomiting and nausea were found to be the two most distressing side-effects. They have an impact on quality of life and compliance with treatment. The development of 5HT3 antagonists has been a major step forward in the prevention and treatment of chemotherapy-induced nausea and vomiting. Presently, these antiemetics are routinely used as concomitant therapy in emetogenic chemotherapy regimens. The purpose of this study was to evaluate the impact of 5HT3 antagonists on patient perceptions of the side-effects of chemotherapy. Coates' survey was replicated in patients who received 5HT3 antagonists for acute nausea and vomiting resulting from emetogenic chemotherapy. Patients received the survey to identify those physical and non-physical side-effects that they attributed to chemotherapy and were asked to rank the five most distressing side-effects. Of the 197 patients who consented to take part in the study, 181 were evaluable. Nausea, hair loss and vomiting were described as the three most distressing side-effects of chemotherapy. Eighty per cent of all the patients actually experienced nausea and 57% experienced vomiting. Hair loss appeared to be more distressing to women (P < 0.001) but, in other aspects, gender, age and marital status did not influence the ranking of the three most distressing side-effects. Constipation was ranked as 6th and was not identified as a distressing side-effect in 1983. Nausea and vomiting remain to be the first and third most distressing side-effects of chemotherapy, even though the incidence and severity of acute nausea and vomiting are now significantly reduced. PMID:9376266

  9. Differences in regional cerebral blood flow response to a 5HT3 antagonist in early- and late-onset cocaine-dependent subjects.

    PubMed

    Adinoff, Bryon; Devous, Michael D; Williams, Mark J; Harris, Thomas S; Best, Susan E; Dong, Hongyun; Zielinski, Tanya

    2014-03-01

    5-hydroxytryptamine 3 (5HT3) receptors are important modulators of mesostriatal dopaminergic transmission and have been implicated in the pathophysiology of cocaine reward, withdrawal and self-administration. In addition, the 5HT3 antagonist ondansetron is effective in treating early-onset, but not late-onset, alcohol-dependent subjects. To explore the role of 5HT3 receptor systems in cocaine addiction using functioning imaging, we administered ondansetron to 23 abstinent, treatment-seeking cocaine-addicted and 22 sex-, age- and race-matched healthy control participants. Differences between early- (first use before 20 years, n = 10) and late-onset (first use after 20 years, n = 10) cocaine-addicted subjects were also assessed. On two separate days, subjects were administered ondansetron (0.15 mg/kg intravenously over 15 minutes) or saline. Regional cerebral blood flow (rCBF) was measured following each infusion with single photon emission computed tomography. No significant rCBF differences between the cocaine-addicted and control participants were observed following ondansetron relative to saline. Early-onset subjects, however, showed increased (P < 0.001) right posterior parahippocampal rCBF following ondansetron. In contrast, late-onset subjects showed decreased rCBF following ondansetron in an overlapping region of the right parahippocampal/hippocampal gyrus. Early-onset subjects also displayed increased rCBF in the left anterior insula and subthalamic nucleus following ondansetron; late-onset subjects showed decreased rCBF in the right anterior insula. These findings suggest that the age of drug use onset is associated with serotonergic biosignatures in cocaine-addicted subjects. Further clarification of these alterations may guide targeted treatment with serotonergic medications similar to those successfully used in alcohol-dependent patients. PMID:22458709

  10. DETERMINATION OF GENOTYPE COMBINATIONS THAT CAN PREDICT THE OUTCOME OF THE TREATMENT OF ALCOHOL DEPENDENCE USING THE 5-HT3 ANTAGONIST ONDANSETRON

    PubMed Central

    Johnson, Bankole A.; Seneviratne, Chamindi; Wang, Xin-Qun; Ait-Daoud, Nassima; Li, Ming D.

    2013-01-01

    Objective Previously, we reported that the 5′-HTTLPR-LL and rs1042173-TT (SLC6A4-LL/TT) genotypes in the serotonin transporter gene predicted a significant reduction in the severity of alcohol consumption among alcoholics receiving the 5-HT3 antagonist ondansetron. In this study, we explored additional markers of ondansetron treatment response in alcoholics by examining polymorphisms in the HTR3A and HTR3B genes, which regulate directly the function and binding of 5-HT3 receptors to ondansetron. Method We genotyped 1 rare and 18 common single-nucleotide polymorphisms in HTR3A and HTR3B in the same sample that we had genotyped for SLC6A4-LL/TT in the previous randomized, double-blind, 11-week clinical trial. Participants were 283 European Americans who received oral ondansetron (4 μg/kg twice daily) or placebo along with weekly cognitive behavioral therapy. Associations of individual and combined genotypes with treatment response on drinking outcomes were analyzed. Results Individuals carrying one or more of genotypes rs1150226-AG and rs1176713-GG in HTR3A and rs17614942-AC in HTR3B showed a significant overall mean difference between ondansetron and placebo in drinks per drinking day (−2.50; effect size (ES)=0.867), percentage of heavy drinking days (−20.58%; ES=0.780), and percentage of days abstinent (18.18%; ES=0.683). Combining these HTR3A/HTR3B and SLC6A4-LL/TT genotypes increased the target cohort from approaching 20% (identified in our previous study) to 34%. Conclusions We present initial evidence suggesting that a combined 5-marker genotype panel can be used to predict the outcome of treatment of alcohol dependence with ondansetron. Additional, larger pharmacogenetic studies would help to validate our results. PMID:23897038

  11. Phenothiazine vs 5HT3 antagonist prophylactic regimens to prevent Post-Anesthesia Care Unit rescue antiemetic: an observational study

    PubMed Central

    Ruiz, Joseph R.; Ensor, Joe E.; Lim, Jeffrey W.; Van Meter, Antoinette; Rahlfs, Thomas F.

    2015-01-01

    Purpose Our practitioners are asked to consider a patient’s postoperative nausea and vomiting (PONV) risk profile when developing their prophylactic antiemetic strategy. There is wide variation in employed strategies, and we have yet to determine the most effective PONV prophylactic regimen. The objective of this study is to compare prophylactic antiemetic regimens containing: phenothiazines to 5HT3 antagonists for effectiveness at reducing the incidence of Post-Anesthesia Care Unit (PACU) rescue antiemetic administration. Methods This is an observational study of 4,392 nonsmoking women who underwent general anesthesia for breast surgery from 1/1/2009 through 6/30/2012. Previous history of PONV or motion sickness (HxPONV/MS) and the use of PACU opioids were recorded. Prophylactic antiemetic therapy was left to the discretion of the anesthesia care team. We compared phenothiazines and 5HT3 antagonists alone and with a glucocorticoid to determine the most effective treatment regimen in our practice for the prevention of the administration of PACU rescue antiemetics. Results Patients who received a phenothiazine regimen compared to a 5HT3 antagonist regimen were less likely to have an antiemetic administered in the PACU (p=0.0100) and this significant difference in rates holds in a logistic regression model adjusted for HxPONV/MS and PACU Opioid use (p=0.0103). Conclusions Based on our findings our clinicians are encouraged to administer a combination of a phenothiazine and a glucocorticoid in female, nonsmoking surgical breast patients for the prevention of PACU rescue antiemetic administration. PMID:26635998

  12. Functional properties of a cloned 5-hydroxytryptamine ionotropic receptor subunit: comparison with native mouse receptors.

    PubMed

    Hussy, N; Lukas, W; Jones, K A

    1994-12-01

    1. A comparative study of the whole-cell and single-channel properties of cloned and native mouse 5-hydroxytryptamine ionotropic receptors (5-HT3) was undertaken using mammalian cell lines expressing the cloned 5-HT3 receptor subunit A (5-HT3R-A), superior cervical ganglia (SCG) neurones and N1E-115 cells. 2. No pharmacological difference was found in the sensitivity to the agonists 5-HT and 2-methyl-5-HT, or to the antagonists d-tubocurare and 3-tropanyl-3,5-dichlorobenzoate (MDL-72222). 3. Current-voltage (I-V) relationships of whole-cell currents showed inward rectification in the three preparations. Rectification was stronger both in cells expressing the 5-HT3R-A subunit and in N1E-115 cells when compared with SCG neurones. 4. No clear openings could be resolved in 5-HT-activated currents in patches excised from cells expressing the 5-HT3R-A subunit or N1E-115 cells. Current fluctuation analysis of whole-cell and excised-patch records revealed a slope conductance of 0.4-0.6 pS in both preparations. Current-voltage relationships of these channels showed strong rectification that fully accounted for the whole-cell voltage dependence. 5. In contrast, single channels of about 10 pS were activated by 5-HT in patches excised from SCG neurones. The weak voltage dependence of their conductance did not account completely for the rectification of whole-cell currents. A lower unitary conductance (3.4 pS) was inferred from whole-cell noise analysis. 6. We conclude that the receptor expressed from the cloned cDNA is indistinguishable from the 5-HT3 receptor of N1E-115 cells, suggesting an identical structure for these two receptors. The higher conductance and different voltage dependence of the 5-HT3 receptor in SCG neurones might indicate the participation of an additional subunit in the structure of native ganglionic 5-HT3 receptors. Homo-oligomeric 5-HT3R-A channels may also be present as suggested by the lower conductance estimated by whole-cell noise analysis. PMID

  13. The reinforcing effects of ethanol within the nucleus accumbens shell involve activation of local GABA and serotonin receptors

    PubMed Central

    Ding, Zheng-Ming; Ingraham, Cynthia M.; Rodd, Zachary A.; McBride, William J.

    2015-01-01

    Ethanol is reinforcing within the nucleus accumbens shell (NACsh), but the underlying mechanisms remain unclear. Ethanol can potentiate the function of the GABAA, GABAB, and 5-HT3 receptors. Therefore, the current study tested the hypothesis that activation of these receptors would be involved in the reinforcing effects of ethanol in the NACsh. An intracranial self-administration (ICSA) procedure was used to assess the reinforcing effects of ethanol in the NACsh of alcohol preferring (P) rats. The ICSA consisted of 7 sessions: 4 sessions to establish 150 mg% ethanol self-infusion into the NACsh; sessions 5 and 6 with co-infusion of ethanol plus one concentration of the GABAA antagonist bicuculline (10 or 100 µM), the GABAB antagonist SCH 50911 (50, 75 or 100 µM), or the 5-HT3 receptor antagonist zacopride (10 or 100 µM); and session 7 with 150 mg% ethanol alone. All groups self-infused ethanol into the NACsh and readily discriminated the active from inactive lever during the acquisition sessions. Co-infusion of 100 µM, but not 10 µM, bicuculline or zacopride significantly decreased active responses during sessions 5 and 6. Co-infusion of 75 µM, but not 50 or 100 µM, SCH 50911 significantly attenuated responses for ethanol. Overall, the results suggest that the reinforcing effects of ethanol in the NACsh may be modulated by activation of local GABAA, GABAB and 5-HT3 receptors. PMID:25922425

  14. Involvement of N-methyl-d-aspartate receptors in the antidepressant-like effect of 5-hydroxytryptamine 3 antagonists in mouse forced swimming test and tail suspension test.

    PubMed

    Kordjazy, Nastaran; Haj-Mirzaian, Arya; Amiri, Shayan; Ostadhadi, Sattar; Amini-Khoei, Hossein; Dehpour, Ahmad Reza

    2016-02-01

    Recent evidence indicates that 5-hydroxytryptamine 3 (5-HT3) antagonists such as ondansetron and tropisetron exert positive behavioral effects in animal models of depression. Due to the ionotropic nature of 5-HT3 and N-methyl-d-aspartate (NMDA) receptors, plus their contribution to the pathophysiology of depression, we investigated the possible role of NMDA receptors in the antidepressant-like effect of 5-HT3 receptor antagonists in male mice. In order to evaluate the animals' behavior in response to different treatments, we performed open-field test (OFT), forced swimming test (FST), and tail-suspension test (TST), which are considered as valid tasks for measuring locomotor activity and depressive-like behaviors in mice. Our data revealed that intraperitoneal (i.p.) administration of tropisetron (5, 10, and 30mg/kg) and ondansetron (0.01, and 0.1μg/kg) significantly decreased the immobility time in FST and TST. Also, co-administration of subeffective doses of tropisetron (1mg/kg, i.p.) or ondansetron (0.001μg/kg, i.p.) with subeffective doses of NMDA receptor antagonists, ketamine (1mg/kg, i.p.), MK-801 (0.05mg/kg, i.p.) and magnesium sulfate (10mg/kg, i.p.) resulted in a reduced immobility time both in FST and TST. The subeffective dose of NMDA (NMDA receptor agonist, 75mg/kg, i.p.) abolished the effects of 5-HT3 antagonists in FST and TST, further supporting the presumed interaction between 5-HT3 and NMDA receptors. These treatments did not affect the locomotor behavior of animals in OFT. Finally, the results of our study suggest that the positive effects of 5-HT3 antagonists on the coping behavior of mice in FST and TST are at least partly mediated through NMDA receptors participation. PMID:26604075

  15. Quercetin inhibits the 5-hydroxytryptamine type 3 receptor-mediated ion current by interacting with pre-transmembrane domain I.

    PubMed

    Lee, Byung-Hwan; Jeong, Sang-Min; Jung, Sang-Min; Lee, Jun-Ho; Kim, Jong-Hoon; Yoon, In-Soo; Lee, Joon-Hee; Choi, Sun-Hye; Lee, Sang-Mok; Chang, Choon-Gon; Kim, Hyung-Chun; Han, YeSun; Paik, Hyun-Dong; Kim, Yangmee; Nah, Seung-Yeol

    2005-08-31

    The flavonoid, quercetin, is a low molecular weight substance found in apple, tomato and other fruit. Besides its antioxidative effect, quercetin, like other flavonoids, has a wide range of neuropharmacological actions including analgesia, and motility, sleep, anticonvulsant, sedative and anxiolytic effects. In the present study, we investigated its effect on mouse 5-hydroxytryptamine type 3 (5-HT3A) receptor channel activity, which is involved in pain transmission, analgesia, vomiting, and mood disorders. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured with the two-electrode voltage clamp technique. In oocytes injected with 5-HT3A receptor cRNA, quercetin inhibited the 5-HT-induced inward peak current (I(5-HT)) with an IC50 of 64.7 +/- 2.2 microM. Inhibition was competitive and voltage-independent. Point mutations of pre-transmembrane domain 1 (pre-TM1) such as R222T and R222A, but not R222D, R222E and R222K, abolished inhibition, indicating that quercetin interacts with the pre-TM1 of the 5-HT3A receptor. PMID:16258243

  16. Differentiated effects of the multimodal antidepressant vortioxetine on sleep architecture: Part 2, pharmacological interactions in rodents suggest a role of serotonin-3 receptor antagonism

    PubMed Central

    Leiser, Steven C; Iglesias-Bregna, Deborah; Westrich, Ligia; Pehrson, Alan L; Sanchez, Connie

    2015-01-01

    Antidepressants often disrupt sleep. Vortioxetine, a multimodal antidepressant acting through serotonin (5-HT) transporter (SERT) inhibition, 5-HT3, 5-HT7 and 5-HT1D receptor antagonism, 5-HT1B receptor partial agonism, and 5-HT1A receptor agonism, had fewer incidences of sleep-related adverse events reported in depressed patients. In the accompanying paper a polysomnographic electroencephalography (sleep-EEG) study of vortioxetine and paroxetine in healthy subjects indicated that at low/intermediate levels of SERT occupancy, vortioxetine affected rapid eye movement (REM) sleep differently than paroxetine. Here we investigated clinically meaningful doses (80–90% SERT occupancy) of vortioxetine and paroxetine on sleep-EEG in rats to further elucidate the serotoninergic receptor mechanisms mediating this difference. Cortical EEG, electromyography (EMG), and locomotion were recorded telemetrically for 10 days, following an acute dose, from rats receiving vortioxetine-infused chow or paroxetine-infused water and respective controls. Sleep stages were manually scored into active wake, quiet wake, and non-REM or REM sleep. Acute paroxetine or vortioxetine delayed REM onset latency (ROL) and decreased REM episodes. After repeated administration, vortioxetine yielded normal sleep-wake rhythms while paroxetine continued to suppress REM. Paroxetine, unlike vortioxetine, increased transitions from non-REM to wake, suggesting fragmented sleep. Next, we investigated the role of 5-HT3 receptors in eliciting these differences. The 5-HT3 receptor antagonist ondansetron significantly reduced paroxetine’s acute effects on ROL, while the 5-HT3 receptor agonist SR57227A significantly increased vortioxetine’s acute effect on ROL. Overall, our data are consistent with the clinical findings that vortioxetine impacts REM sleep differently than paroxetine, and suggests a role for 5-HT3 receptor antagonism in mitigating these differences. PMID:26174134

  17. Differentiated effects of the multimodal antidepressant vortioxetine on sleep architecture: Part 2, pharmacological interactions in rodents suggest a role of serotonin-3 receptor antagonism.

    PubMed

    Leiser, Steven C; Iglesias-Bregna, Deborah; Westrich, Ligia; Pehrson, Alan L; Sanchez, Connie

    2015-10-01

    Antidepressants often disrupt sleep. Vortioxetine, a multimodal antidepressant acting through serotonin (5-HT) transporter (SERT) inhibition, 5-HT3, 5-HT7 and 5-HT1D receptor antagonism, 5-HT1B receptor partial agonism, and 5-HT1A receptor agonism, had fewer incidences of sleep-related adverse events reported in depressed patients. In the accompanying paper a polysomnographic electroencephalography (sleep-EEG) study of vortioxetine and paroxetine in healthy subjects indicated that at low/intermediate levels of SERT occupancy, vortioxetine affected rapid eye movement (REM) sleep differently than paroxetine. Here we investigated clinically meaningful doses (80-90% SERT occupancy) of vortioxetine and paroxetine on sleep-EEG in rats to further elucidate the serotoninergic receptor mechanisms mediating this difference. Cortical EEG, electromyography (EMG), and locomotion were recorded telemetrically for 10 days, following an acute dose, from rats receiving vortioxetine-infused chow or paroxetine-infused water and respective controls. Sleep stages were manually scored into active wake, quiet wake, and non-REM or REM sleep. Acute paroxetine or vortioxetine delayed REM onset latency (ROL) and decreased REM episodes. After repeated administration, vortioxetine yielded normal sleep-wake rhythms while paroxetine continued to suppress REM. Paroxetine, unlike vortioxetine, increased transitions from non-REM to wake, suggesting fragmented sleep. Next, we investigated the role of 5-HT3 receptors in eliciting these differences. The 5-HT3 receptor antagonist ondansetron significantly reduced paroxetine's acute effects on ROL, while the 5-HT3 receptor agonist SR57227A significantly increased vortioxetine's acute effect on ROL. Overall, our data are consistent with the clinical findings that vortioxetine impacts REM sleep differently than paroxetine, and suggests a role for 5-HT3 receptor antagonism in mitigating these differences. PMID:26174134

  18. SPINAL CORD MECHANISMS MEDIATING BEHAVIORAL HYPERALGESIA INDUCED BY NEUROKININ-1 TACHYKININ RECEPTOR ACTIVATION IN THE ROSTRAL VENTROMEDIAL MEDULLA

    PubMed Central

    Lagraize, S. C.; Guo, W.; Yang, K.; Wei, F.; Ren, K.; Dubner, R.

    2010-01-01

    Hyperalgesia in animal injury models is linked to activation of descending raphespinal modulatory circuits originating in the rostral ventromedial medulla (RVM). A neurokinin-1 (NK-1) receptor antagonist microinjected into the RVM before or after inflammation produced by complete Freund’s adjuvant (CFA) resulted in an attenuation of thermal hyperalgesia. A transient (acute) or a continuous infusion of Substance P (SP) microinjected into the RVM of non-inflamed animals led to similar pain hypersensitivity. Intrathecal pretreatment or post-treatment of a 5-HT3 receptor antagonist (Y-25130 or ondansetron) blocked the SP-induced hyperalgesia. The SP-induced hyperalgesia was both GABAA and NMDA receptor-dependent after pre- and post-treatment with selective antagonists at the spinal level. A microinjection of SP into the RVM also led to increased NMDA NR1 receptor subunit phosphorylation in spinal cord tissue. The GABAA receptor-mediated hyperalgesia involved a shift in the anionic gradient in dorsal horn nociceptive neurons and an increase in phosphorylated NKCC1 protein (isoform of the Na-K-Cl cotransporter). Following a low dose of SP infused into the RVM, intrathecal muscimol (GABAA agonist) increased SP-induced thermal hyperalgesia, phosphorylated NKCC1 protein expression, and NMDA NR1 subunit phosphorylation in the spinal cord. The thermal hyperalgesia was blocked by intrathecal gabazine, the GABAA receptor antagonist, and MK-801, the NMDA receptor channel blocker. These findings indicate that NK-1 receptors in the RVM are involved in SP-induced thermal hyperalgesia, this hyperalgesia is 5-HT3-receptor dependent at the spinal level, and involves the functional interaction of spinal GABAA and NMDA receptors. PMID:20888891

  19. Pharmacological characterisation of strychnine and brucine analogues at glycine and alpha7 nicotinic acetylcholine receptors.

    PubMed

    Jensen, Anders A; Gharagozloo, Parviz; Birdsall, Nigel J M; Zlotos, Darius P

    2006-06-01

    Strychnine and brucine from the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors, including some members of the superfamily of ligand-gated ion channels. In this study, we have characterised the pharmacological properties of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human alpha1 and alpha1beta glycine receptors and at a chimera consisting of the amino-terminal domain of the alpha7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion channel domain of the 5-HT3A serotonin receptor. Although the majority of the analogues displayed significantly increased Ki values at the glycine receptors compared to strychnine and brucine, a few retained the high antagonist potencies of the parent compounds. However, mirroring the pharmacological profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an insight into the structure-activity relationships for strychnine and brucine analogues at these ligand-gated ion channels. PMID:16687139

  20. Vortioxetine, a novel antidepressant with multimodal activity: review of preclinical and clinical data.

    PubMed

    Sanchez, Connie; Asin, Karen E; Artigas, Francesc

    2015-01-01

    Vortioxetine, a novel antidepressant for the treatment of major depressive disorder (MDD), is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist and serotonin (5-HT) transporter (SERT) inhibitor. Here we review its preclinical and clinical properties and discuss translational aspects. Vortioxetine increases serotonergic, noradrenergic, dopaminergic, cholinergic, histaminergic and glutamatergic neurotransmission in brain structures associated with MDD. These multiple effects likely derive from its interaction with 5-HT-receptor-mediated negative feedback mechanisms controlling neuronal activity. In particular, 5-HT3 receptors may play a prominent role, since their blockade i) increases pyramidal neuron activity by removing 5-HT3 receptor-mediated excitation of GABA interneurons, and ii) augments SSRI effects on extracellular 5-HT. However, modulation of the other 5-HT receptor subtypes also likely contributes to vortioxetine's pharmacological effects. Preclinical animal models reveal differences from SSRIs and SNRIs, including antidepressant-like activity, increased synaptic plasticity and improved cognitive function. Vortioxetine had clinical efficacy in patients with MDD: 11 placebo-controlled studies (including one in elderly) with efficacy in 8 (7 positive, 1 supportive), 1 positive active comparator study plus a positive relapse prevention study. In two positive studies, vortioxetine was superior to placebo in pre-defined cognitive outcome measures. The clinically effective dose range (5-20mg/day) spans ~50 to >80% SERT occupancy. SERT and 5-HT3 receptors are primarily occupied at 5mg, while at 20mg, all targets are likely occupied at functionally relevant levels. The side-effect profile is similar to that of SSRIs, with gastrointestinal symptoms being most common, and a low incidence of sexual dysfunction and sleep disruption possibly ascribed to vortioxetine's receptor modulation. PMID:25016186

  1. Dopamine D2/3 receptor antagonism reduces activity-based anorexia.

    PubMed

    Klenotich, S J; Ho, E V; McMurray, M S; Server, C H; Dulawa, S C

    2015-01-01

    Anorexia nervosa (AN) is an eating disorder characterized by severe hypophagia and weight loss, and an intense fear of weight gain. Activity-based anorexia (ABA) refers to the weight loss, hypophagia and paradoxical hyperactivity that develops in rodents exposed to running wheels and restricted food access, and provides a model for aspects of AN. The atypical antipsychotic olanzapine was recently shown to reduce both AN symptoms and ABA. We examined which component of the complex pharmacological profile of olanzapine reduces ABA. Mice received 5-HT(2A/2C), 5-HT3, dopamine D1-like, D2, D3 or D2/3 antagonist treatment, and were assessed for food intake, body weight, wheel running and survival in ABA. D2/3 receptor antagonists eticlopride and amisulpride reduced weight loss and hypophagia, and increased survival during ABA. Furthermore, amisulpride produced larger reductions in weight loss and hypophagia than olanzapine. Treatment with either D3 receptor antagonist SB277011A or D2 receptor antagonist L-741,626 also increased survival. All the other treatments either had no effect or worsened ABA. Overall, selective antagonism of D2 and/or D3 receptors robustly reduces ABA. Studies investigating the mechanisms by which D2 and/or D3 receptors regulate ABA, and the efficacy for D2/3 and/or D3 antagonists to treat AN, are warranted. PMID:26241351

  2. Dopamine D2/3 receptor antagonism reduces activity-based anorexia

    PubMed Central

    Klenotich, S J; Ho, E V; McMurray, M S; Server, C H; Dulawa, S C

    2015-01-01

    Anorexia nervosa (AN) is an eating disorder characterized by severe hypophagia and weight loss, and an intense fear of weight gain. Activity-based anorexia (ABA) refers to the weight loss, hypophagia and paradoxical hyperactivity that develops in rodents exposed to running wheels and restricted food access, and provides a model for aspects of AN. The atypical antipsychotic olanzapine was recently shown to reduce both AN symptoms and ABA. We examined which component of the complex pharmacological profile of olanzapine reduces ABA. Mice received 5-HT2A/2C, 5-HT3, dopamine D1-like, D2, D3 or D2/3 antagonist treatment, and were assessed for food intake, body weight, wheel running and survival in ABA. D2/3 receptor antagonists eticlopride and amisulpride reduced weight loss and hypophagia, and increased survival during ABA. Furthermore, amisulpride produced larger reductions in weight loss and hypophagia than olanzapine. Treatment with either D3 receptor antagonist SB277011A or D2 receptor antagonist L-741,626 also increased survival. All the other treatments either had no effect or worsened ABA. Overall, selective antagonism of D2 and/or D3 receptors robustly reduces ABA. Studies investigating the mechanisms by which D2 and/or D3 receptors regulate ABA, and the efficacy for D2/3 and/or D3 antagonists to treat AN, are warranted. PMID:26241351

  3. Involvement of 5-HT₃ and 5-HT₄ receptors in the regulation of circadian clock gene expression in mouse small intestine.

    PubMed

    Aoki, Natsumi; Watanabe, Hiroyuki; Okada, Kazuya; Aoki, Kazuyuki; Imanishi, Takuma; Yoshida, Daisuke; Ishikawa, Ryosuke; Shibata, Shigenobu

    2014-01-01

    Several lines of evidence suggest that 5-HT receptors play a critical role in the expression of clock genes in the suprachiasmatic nucleus, the main circadian oscillator in hamsters. The contributions of 5-HT-receptor subtypes in the intestine, where they are expressed at high concentrations, are however not yet clarified. The 5-HT synthesis inhibitor, p-chlorophenylalanine, attenuated the daily rhythm of Per1 and Per2 gene expression in the intestine. Injection of 5-HT and agonists of the 5-HT3 and 5-HT4 receptors increased Per1/Per2 expression and decreased Bmal1 expression in a dose-dependent manner. Although treatment with antagonists of 5-HT3 and 5-HT4 alone did not affect clock gene expression, co-injection of these antagonists with 5-HT blocked the 5-HT-induced changes in clock gene expression. Increased tissue levels of 5-HT due to treatment with the antidepressants clomipramine and fluvoxamine did not affect clock gene expression. The present results suggest that the 5-HT system in the small intestine may play a critical role in regulating circadian rhythms through 5-HT3/5-HT4-receptor activation. PMID:24492464

  4. Synergistic effect of 5-HT1A and σ1 receptor activation on prefrontal dopaminergic transmission under circulating steroid deficiency.

    PubMed

    Hiramatsu, Naoki; Ago, Yukio; Hasebe, Shigeru; Nishimura, Akira; Mori, Kazuya; Takuma, Kazuhiro; Matsuda, Toshio

    2013-12-01

    Serotonin (5-HT)1A and σ1 receptors have been implicated in psychiatric disorders. We previously found that combined 5-HT reuptake inhibition and σ1 receptor activation has a synergistic effect on prefrontal dopaminergic transmission in adrenalectomized/castrated mice lacking circulating steroid hormones. In the present study, we examined the mechanisms underlying this neurochemical synergism. Systemic administration of fluvoxamine, a selective 5-HT reuptake inhibitor with agonistic activity towards the σ1 receptor, increased prefrontal dopamine (DA) levels, and adrenalectomy/castration potentiated this fluvoxamine-induced increase in DA. This enhancement of DA release was blocked by WAY100635 (a 5-HT1A receptor antagonist), but not by ritanserin (a 5-HT2 receptor antagonist), azasetron (a 5-HT3 receptor antagonist) or SB269970 (a 5-HT7 receptor antagonist). Individually, osemozotan (a 5-HT1A receptor agonist) and (+)-SKF-10,047 (a σ1 receptor agonist) did not alter prefrontal monoamine levels in adrenalectomized/castrated and sham-operated mice differentially. In contrast, co-administration of these drugs increased prefrontal DA levels to a greater extent in adrenalectomized/castrated mice than in sham-operated animals. Furthermore, co-administration of osemozotan and (+)-SKF-10,047 increased expression of the neuronal activity marker c-Fos in the ventral tegmental area of adrenalectomized/castrated mice, but not in sham-operated animals. These findings suggest that combined activation of 5-HT1A and σ1 receptors has a synergistic effect on prefrontal dopaminergic transmission under circulating steroid deficiency, and that this interaction may play an important role in the regulation of the prefrontal DA system. PMID:23851260

  5. Serotonergic receptor mechanisms underlying antidepressant-like action in the progesterone withdrawal model of hormonally induced depression in rats.

    PubMed

    Li, Yan; Raaby, Kasper F; Sánchez, Connie; Gulinello, Maria

    2013-11-01

    Hormonally induced mood disorders such as premenstrual dysphoric disorder (PMDD) are characterized by a range of physical and affective symptoms including anxiety, irritability, anhedonia, social withdrawal and depression. Studies demonstrated rodent models of progesterone withdrawal (PWD) have a high level of constructive and descriptive validity to model hormonally-induced mood disorders in women. Here we evaluate the effects of several classes of antidepressants in PWD female Long-Evans rats using the forced swim test (FST) as a measure of antidepressant activity. The study included fluoxetine, duloxetine, amitriptyline and an investigational multimodal antidepressant, vortioxetine (5-HT(3), 5-HT(7) and 5-HT(1D) receptor antagonist; 5-HT(1B) receptor partial agonist; 5-HT(1A) receptor agonist; inhibitor of the serotonin transporter (SERT)). After 14 days of administration, amitriptyline and vortioxetine significantly reduced immobility in the FST whereas fluoxetine and duloxetine were ineffective. After 3 injections over 48 h, neither fluoxetine nor duloxetine reduced immobility, whereas amitriptyline and vortioxetine significantly reduced FST immobility during PWD. When administered acutely during PWD, the 5-HT(1A) receptor agonist, flesinoxan, significantly reduced immobility, whereas the 5-HT(1A) receptor antagonist, WAY-100635, increased immobility. The 5-HT(3) receptor antagonist, ondansetron, significantly reduced immobility, whereas the 5-HT(3) receptor agonist, SR-57227, increased immobility. The 5-HT(7) receptor antagonist, SB-269970, was inactive, although the 5-HT(7) receptor agonist, AS-19, significantly increased PWD-induced immobility. None of the compounds investigated (ondansetron, flesinoxan and SB-269970) improved the effect of fluoxetine during PWD. These data indicate that modulation of specific 5-HT receptor subtypes is critical for manipulating FST immobility in this model of hormone-induced depression. PMID:24016840

  6. Effects of OCT1 polymorphisms on the cellular uptake, plasma concentrations and efficacy of the 5-HT(3) antagonists tropisetron and ondansetron.

    PubMed

    Tzvetkov, M V; Saadatmand, A R; Bokelmann, K; Meineke, I; Kaiser, R; Brockmöller, J

    2012-02-01

    After uptake into liver cells, the antiemetic drugs tropisetron and ondansetron undergo metabolic inactivation by cytochrome P450 2D6 (CYP2D6). We investigated whether the hepatic organic cation transporter 1 (OCT1; SLC22A1) mediates cellular uptake and whether common OCT1 loss-of-function polymorphisms affect pharmacokinetics and efficacy of both drugs. Both tropisetron and ondansetron inhibited ASP(+) uptake in OCT1-overexpressing HEK293 cells. Overexpression of wild-type, but not OCT1 loss-of-function variants, significantly increased tropisetron uptake. Correspondingly, patients with two loss-of-function OCT1 alleles had higher tropisetron plasma concentrations (n=59, P<0.04) and higher clinical efficacy (n=91, P=0.009) compared with carriers of fully active OCT1. Overexpression of OCT1 did not increase ondansetron uptake. Nevertheless, OCT1 genotypes correlated with pharmacokinetics (n=45, P<0.05) and clinical efficacy (n=222, P<0.02) of ondansetron, the effect size of OCT1 genotypes on pharmacokinetics and efficacy was greater for tropisetron than for ondansetron. In conclusion, in addition to the known effects of CYP2D6, OCT1 deficiency may increase efficacy of tropisetron and potentially of ondansetron by limiting their hepatic uptake. PMID:20921968

  7. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  8. The metabolism of the 5HT3 antagonists, ondansetron, alosetron and GR87442 II: investigation into the in vitro methods used to predict the in vivo hepatic clearance of ondansetron, alosetron and GR87442 in the rat, dog and human.

    PubMed

    Somers, G I; Bayliss, M K; Houston, J B

    2007-08-01

    The in vitro clearances of the 5HT3 antagonists, ondansetron, alosetron and GR87442 were investigated. Intrinsic clearances using either metabolite formation or substrate depletion methods were equivalent (R2 = 0.95). Hepatocytes from preclinical species were superior to microsomes for the prediction of hepatic clearance (CL(H)), whereas the predictions from human microsomes and hepatocytes were similar. Using a non-restrictive model, seven of the nine CL(H) predictions using hepatocytes were within 2-fold of the in vivo CL(H) values. If the unbound fraction was included, the clearance of the compounds was generally under-predicted by both in vitro models. However, for the most metabolically stable compound, GR87442, the non-restrictive model over-predicted CLp. This and the possibility of extrahepatic metabolism indicate that the restrictive model is more appropriate for prediction of CL(H). The rank order of metabolic stability correlated with that in vivo. All three compounds were more metabolically stable in human than in the preclinical animal species examined. PMID:17701833

  9. [Molecular physiology of glycine receptors in nervous system of vertebrates].

    PubMed

    2014-03-01

    Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. It has been cloned one beta and four alpha subunits of glycine receptor, which are specifically distributed in different areas of the nervous system. Due to their specific molecular properties and distribution, different subunits ensure important physiological functions: from control of motor activity and regulation of neuronal differentiation to sensory information processing and modulation of pain sensitivity. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their modulation and dysfunction. PMID:25508361

  10. [Molecular physiology of glycine receptors in nervous system of vertebrates].

    PubMed

    Maleeva, G V; Brezhestovskiĭ, P D

    2014-03-01

    Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. It has been cloned one beta and four alpha subunits of glycine receptor, which are specifically distributed in different areas of the nervous system. Due to their specific molecular properties and distribution, different subunits ensure important physiological functions: from control of motor activity and regulation of neuronal differentiation to sensory information processing and modulation of pain sensitivity. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their modulation and dysfunction. PMID:25464730

  11. Direct and Indirect 5-HT receptor agonists produce gender-specific effects on locomotor and vertical activity in C57 BL/6J mice

    PubMed Central

    Brookshire, Bethany R.; Jones, Sara R.

    2009-01-01

    It is well established that the dopamine (DA) and serotonin (5-HT) systems have extensive and complex interactions. However, the effects of specific 5-HT receptor agonists on traditionally DA-related behaviors remain unclear. Our goal in these studies was to characterize the effects of 5-HT receptor agonists on measures of locomotor activity and vertical rearing. The SSRIs fluoxetine and citalopram produced significant decreases in locomotor activity and vertical rearing at the highest doses used with females significant more sensitive to citalopram. The 5-HT1A agonist 8-OH-DPAT and the 5-HT2C agonist MK 212 significantly decreased activity in both male and female mice, with females more sensitive to 8-OH-DPAT. In contrast, the 5-HT1B agonist RU 24969 and the 5-HT2A agonist DOI both increased activity, with DOI exhibiting differential effects with regard to sex. Finally, the 5-HT3 agonist SR 57227 produced significant locomotor increases only in female mice at the lowest dose. The results of these experiments define locomotor profiles of several 5-HT agonists in male and female C57BL/6J mice, providing a foundation for further explorations of 5-HT receptor effects on activity. PMID:19698737

  12. Review article: serotonin receptors and transporters -- roles in normal and abnormal gastrointestinal motility.

    PubMed

    Gershon, M D

    2004-11-01

    The gut is the only organ that can display reflexes and integrative neuronal activity even when isolated from the central nervous system. This activity can be triggered by luminal stimuli that are detected by nerves via epithelial intermediation. Epithelial enterochromaffin cells act as sensory transducers that activate the mucosal processes of both intrinsic and extrinsic primary afferent neurones through their release of 5-hydroxytryptamine (5-HT). Intrinsic primary afferent neurones are present in both the submucosal and myenteric plexuses. Peristaltic and secretory reflexes are initiated by submucosal intrinsic primary afferent neurones, which are stimulated by 5-HT acting at 5-HT(1P) receptors. 5-HT acting at 5-HT4 receptors enhances the release of transmitters from their terminals and from other terminals in prokinetic reflex pathways. Signalling to the central nervous system is predominantly 5-HT3 mediated, although serotonergic transmission within the enteric nervous system and the activation of myenteric intrinsic primary afferent neurones are also 5-HT3 mediated. The differential distribution of 5-HT receptor subtypes makes it possible to use 5-HT3 antagonists and 5-HT4 agonists to treat intestinal discomfort and motility. 5-HT3 antagonists alleviate the nausea and vomiting associated with cancer chemotherapy and the discomfort from the bowel in irritable bowel syndrome; however, because 5-HT-mediated fast neurotransmission within the enteric nervous system and the stimulation of mucosal processes of myenteric intrinsic primary afferent neurones are 5-HT3 mediated, 5-HT3 antagonists tend to be constipating and should be used only when pre-existing constipation is not a significant component of the problem to be treated. In contrast, 5-HT4 agonists, such as tegaserod, are safe and effective in the treatment of irritable bowel syndrome with constipation and chronic constipation. They do not stimulate nociceptive extrinsic nerves nor initiate peristaltic and

  13. Phosphoinositide system-linked serotonin receptor subtypes and their pharmacological properties and clinical correlates.

    PubMed Central

    Pandey, S C; Davis, J M; Pandey, G N

    1995-01-01

    Serotonergic neurotransmission represents a complex mechanism involving pre- and post-synaptic events and distinct 5-HT receptor subtypes. Serotonin (5-HT) receptors have been classified into several categories, and they are termed as 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7 type receptors. 5-HT1 receptors have been further subdivided into 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E and 5-HT1F. 5-HT2 receptors have been divided into 5-HT2A, 5-HT2B and 5-HT2C receptors. All 5-HT2 receptor subtypes are linked to the multifunctional phosphoinositide (PI) signalling system. 5-HT3 receptors are considered ion-gated receptors and are also linked to the PI signalling system by an unknown mechanism. The 5-HT2A receptor subtype is the most widely studied of the 5-HT receptors in psychiatric disorders (for example, suicide, depression and schizophrenia) as well as in relation to the mechanism of action of antidepressant drugs. The roles of 5-HT2C and 5-HT3 receptors in psychiatric disorders are less clear. These 5-HT receptors also play an important role in alcoholism. It has been shown that 5-HT2A, 5-HT2C and 5-HT3 antagonists cause attenuation of alcohol intake in animals and humans. However, the exact mechanisms are unknown. The recent cloning of the cDNAs for 5-HT2A, 5-HT2C and 5-HT3 receptors provides the opportunity to explore the molecular mechanisms responsible for the alterations in these receptors during illness as well as pharmacotherapy. This review article will focus on the current research into the pharmacological properties, molecular biology, and clinical correlates of 5-HT2A, 5-HT2C and 5-HT3 receptors. PMID:7786883

  14. GABA-A Receptor Inhibition of Local Calcium Signaling in Spines and Dendrites

    PubMed Central

    Marlin, Joseph J.

    2014-01-01

    Cortical interneurons activate GABA-A receptors to rapidly control electrical and biochemical signaling at pyramidal neurons. Different populations of interneurons are known to uniquely target the soma and dendrites of pyramidal neurons. However, the ability of these interneurons to inhibit Ca2+ signaling at spines and dendrites is largely unexplored. Here we use whole-cell recordings, two-photon microscopy, GABA uncaging and optogenetics to study dendritic inhibition at layer 5 (L5) pyramidal neurons in slices of mouse PFC. We first show that GABA-A receptors strongly inhibit action potential (AP)-evoked Ca2+ signals at both spines and dendrites. We find robust inhibition over tens of milliseconds that spreads along the dendritic branch. However, we observe no difference in the amount of inhibition at neighboring spines and dendrites. We then examine the influence of interneurons expressing parvalbumin (PV), somatostatin (SOM), or 5HT3a receptors. We determine that these populations of interneurons make unique contacts onto the apical and basal dendrites of L5 pyramidal neurons. We also show that SOM and 5HT3a but not PV interneurons potently inhibit AP Ca2+ signals via GABA-A receptors at both spines and dendrites. These findings reveal how multiple interneurons regulate local Ca2+ signaling in pyramidal neurons, with implications for cortical function and disease. PMID:25429132

  15. Serotonin receptors are selectively expressed in the avian germ cells and early embryos.

    PubMed

    Stępińska, Urszula; Kuwana, Takashi; Olszańska, Bożenna

    2015-06-01

    The expression of nine serotonin (5-HT) receptor transcripts was studied using reverse transcription polymerase chain reaction (RT-PCR) in germ cells, cleavage and gastrulation stages of Japanese quail, and qPCR for 5-HT3 and 5-HT4 receptors in oocytes and embryos. We show the presence/absence of nine serotonin transcripts known in birds for receptors 5-HT1A, 5-HT1F, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT6 and 5-HT7A in avian germ cells and early embryos. The absence of 5-HT3 and 5-HT5A in primordial germ cells and of 5-HT3 and 5-HT7A in sperm is characteristic. All transcripts appeared in oocytes at all stages (except for 5-HT3 and 5-HT5A transcripts) and all were present in cleaving embryos and at gastrulation, except for 5-HT3, which was permanently observed as late as in stage 4. Interestingly, 5-HT3 and 5-HT5A receptors accumulated in 3-mm and F1 oocytes but were degraded at ovulation and started to be re-transcribed in cleavage stage II embryos and beyond. The selective appearance of 5-HT receptors in germ cells and early embryos supports the hypothesis that serotonin may act as a signalling molecule at early stages of germ line and embryo differentiation via individual receptors present during different stages, when specialized communication systems are not yet developed. PMID:24521994

  16. Characterizing new fluorescent tools for studying 5-HT₃ receptor pharmacology.

    PubMed

    Jack, Thomas; Simonin, Jonathan; Ruepp, Marc-David; Thompson, Andrew J; Gertsch, Jürg; Lochner, Martin

    2015-03-01

    The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues. PMID:25460187

  17. Intracolonical administration of protease-activated receptor-2 agonists produced visceral hyperalgesia by up-regulating serotonin in the colon of rats.

    PubMed

    Li, Zhi; Zhang, Xiao-Jun; Xu, Hong-xi; Sung, Joseph J Y; Bian, Zhao-xiang

    2009-03-15

    This study aimed to investigate the underlying mechanism of protease-activated receptor-2 (PAR-2) agonist-induced visceral hyperalgesia. Male Sprague-Dawley rat pups were submitted to colonic injection of PAR-2 agonist for 6 consecutive days. The visceral sensitivity to colorectal distention was evaluated by electromyography. The enterochromaffin (EC) cell number, 5-HT content and tryrptophan hydroxylase (TPH) protein expression were detected with immunohistochemistry, fluorescent measurement and Western blot analysis. PAR-2 agonist induced a significant increase of visceral nociceptive response to colorectal distention and a series of neurochemical changes in rat colon, including proliferation of EC cells, increased 5-HT content and enhanced TPH expression. Expression of PAR-2 in EC cells was reported for the first time. Further, selective 5-HT(3) receptor antagonist alosteron significantly inhibited PAR-2-induced visceral hyperalgesia. The enhanced 5-HT signaling is likely responsible for the visceral hyperalgesia induced by PAR-2 agonist. Interruption of this pathway is a possible target for the treatment of visceral hyperalgesia in gastrointestinal diseases. PMID:19374846

  18. Cloning and expression of ligand-gated ion-channel receptor L2 in central nervous system

    SciTech Connect

    Houtani, Takeshi; Munemoto, Yumi; Kase, Masahiko; Sakuma, Satoru; Tsutsumi, Toshiyuki; Sugimoto, Tetsuo . E-mail: sugimoto@takii.kmu.ac.jp

    2005-09-23

    An orphan receptor of ligand-gated ion-channel type (L2, also termed ZAC according to the presence of zinc ion for channel activation) was identified by computer-assisted search programs on human genome database. The L2 protein shares partial homology with serotonin receptors 5HT3A and 5HT3B. We have cloned L2 cDNA derived from human caudate nucleus and characterized the exon-intron structure as follows: (1) The L2 protein has four transmembrane regions (M1-M4) and a long cytoplasmic loop between M3 and M4. (2) The sequence is conserved in species including chimpanzee, dog, cow, and opossum. (3) Nine exons form its protein-coding region and especially exon 5 corresponds to a disulfide bond region on the amino-terminal side. Our analysis using multiple tissue cDNA panels revealed that at least two splicing variants of L2 mRNA are present. The cDNA PCR amplification study revealed that L2 mRNA is expressed in tissues including brain, pancreas, liver, lung, heart, kidney, and skeletal muscle while 5HT3A mRNA could be detected in brain, heart, placenta, lung, kidney, pancreas, and skeletal muscle, and 5HT3B mRNA in brain, kidney, and skeletal muscle, suggesting different significance in tissue expression of these receptors. Regional expression of L2 mRNA and protein was examined in brain. The RT-PCR studies confirmed L2 mRNA expression in hippocampus, striatum, amygdala, and thalamus in adult brain. The L2 protein was immunolocalized by using antipeptide antibodies. Immunostained tissue sections revealed that L2-like immunoreactivity was dominantly expressed in the hippocampal CA3 pyramidal cells and in the polymorphic layer of the dentate gyrus. We analyzed the expression of L2 protein in HEK293 cells using GFP fusion protein reporter system. Western blots revealed that L2 protein confers sugar chains on the extracellular side. In transfected HEK293 cells, cellular membranes and intracellular puncta were densely labeled with GFP, suggesting selective dispatch to the

  19. Mechanism for the activation of glutamate receptors

    Cancer.gov

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  20. Expression of Serotonin Receptors in the Colonic Tissue of Chronic Diarrhea Rats

    PubMed Central

    Zhu, Tong; Qiu, Juanjuan; Wan, Jiajia; Wang, Fengyun; Tang, Xudong; Guo, Huishu

    2016-01-01

    Background/Aims: This study aimed to investigate the difference among the expression of serotonin receptors (5-HT3, 5-HT4, and 5-HT7 receptors) in colonic tissue of chronic diarrhea rats. Materials and Methods: A rat model of chronic diarrhea was established by lactose diet. The expression of 5-HT3, 5-HT4, and 5-HT7 receptors in the colonic tissue was detected using immunohistochemistry, real-time PCR and Western blotting techniques. Results: There is no significant difference on the protein expression of 5-HT3 receptor between the normal group and the chronic diarrhea model group. The mRNA expression of 5-HT3 receptor in the chronic diarrhea model group was significantly lower than that in the normal group (n = 10; P < 0.01). The protein and mRNA expression of 5-HT4 receptor in the chronic diarrhea model group were significantly higher than those in the normal group (n = 10; P < 0.05, P < 0.01). On the contrary, the protein and mRNA expressions of 5-HT7 receptor in the chronic diarrhea model group were significantly decreased compared with the normal group (n = 10; P < 0.01, P < 0.01). Conclusions: The results suggested the receptors of 5-HT4 and 5-HT7 may be involved in inducing diarrhea by lactose diet. PMID:27184643

  1. Paired inhibitory and activating receptor signals.

    PubMed

    Taylor, L S; Paul, S P; McVicar, D W

    2000-01-01

    The immunological literature has become inundated with reports regarding paired inhibitory receptors. Paired inhibitory receptor systems are highly conserved families that contain receptors involved in either cellular inhibition or activation. In most cases the paired putative biochemical antagonists are co-expressed on a given cell and thought to bind similar, if not identical, ligands making their biological role difficult to understand. Examples of these systems include immunoglobulin (Ig)-like receptors (Killer Ig Receptors, Immunoglobulin-like Transcripts/Leukocyte Ig-like Receptors/Monocyte Macrophage Ig Receptors, and Paired Ig-like Receptors), and type II lectin-like receptor systems (NKG2 and Ly49). General characteristics of these inhibitory receptors include a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is phosphorylated upon engagement and recruits protein tyrosine phosphatases that dephosphorylate cellular substrates that would otherwise mediate activation. In contrast, the activating receptors of these pairs use charged residues within their transmembrane domains to associate with various signal transduction chains including the gamma chain of the receptor for the Fc portion of IgE, DAP12 or DAP10. Once phosphorylated, these chains direct the signal transduction cascade resulting in cellular activation. Here we review the signaling of several paired systems and present the current models for their signal transduction cascades. PMID:11258418

  2. The 5-HT4 receptor: molecular cloning and pharmacological characterization of two splice variants.

    PubMed Central

    Gerald, C; Adham, N; Kao, H T; Olsen, M A; Laz, T M; Schechter, L E; Bard, J A; Vaysse, P J; Hartig, P R; Branchek, T A

    1995-01-01

    Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable. Images PMID:7796807

  3. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2015-07-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism. PMID:25910812

  4. Hormone activation of baculovirus expressed progesterone receptors.

    PubMed

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  5. Mechanisms and latest clinical studies of new NK1 receptor antagonists for chemotherapy-induced nausea and vomiting: Rolapitant and NEPA (netupitant/palonosetron).

    PubMed

    Rojas, Camilo; Slusher, Barbara S

    2015-12-01

    Many patients undergoing moderately or highly emetogenic chemotherapy experience chemotherapy-induced nausea/vomiting (CINV) and report reduced daily functioning, despite prophylaxis with antiemetic drugs. While modern antiemetics have largely alleviated acute emesis, management of nausea and delayed emesis remains particularly challenging. We briefly review the pathophysiologic mechanisms of CINV and the clinical impact of current antiemetics, i.e., the serotonin subtype 3 (5-HT3) receptor antagonists (RAs) and neurokinin-1 (NK1)RAs, before summarizing recent data from clinical trials of new agents. The new antiemetics reviewed include the two most recently approved drugs, the NK1RA rolapitant and the fixed-dose combination product, NEPA, which is composed of the NK1RA netupitant and the 5-HT3RA palonosetron. Phase 3 studies demonstrate improved control of CINV in the delayed and overall phases when rolapitant is added to a standard 5-HT3RA regimen. Phase 2 and phase 3 clinical trials with NEPA demonstrate improved control of CINV in the acute, delayed, and overall phases vs. 5-HT3RA regimens. These data suggest that delayed emesis can be substantially reduced via combined 5-HT3 and NK1 receptor neurotransmitter pathway inhibition. PMID:26442475

  6. Neuronal nicotinic acetylcholine receptor modulation by general anesthetics.

    PubMed

    Flood, P; Role, L W

    1998-11-23

    1. General anesthetics have been shown to inhibit synaptic transmission in multiple areas of the central and peripheral nervous systems. 2. The mechanism of inhibition is not well understood. 3. It has become clear that general anesthetics modulate the function of members of the ligand gated ion channel superfamily, including receptors for GABA(A), glycine (Harrison et al., Mol. Pharmacol. 44(3), 1993, 628-632) and 5HT3 (Zhou and Lovinger, J. Pharmacol. Exp. Therap. 278(2), 1996, 732-740). 4. Studies of the activity of general anesthetics on recombinant neuronal nicotinic acetylcholine receptors have added this receptor family to those potently inhibited by general anesthetics (Flood et al., Anesthesiology 86(4), 1997, 859-865; Violet et al., Anesthesiology 86(4), 1997, 866-874). 5. Studies of neuronal nicotinic receptors in native neurons suggest that the inhibition of these receptors by general anesthetics at low clinical concentrations may be biologically significant (Nicoll, Science 199(4327), 1978, 451-452). 6. Recent work on neuronal nicotinic acetylcholine receptors in the central nervous system suggests that their primary role may be to modulate synaptic transmission (Role and Berg, Neuron 16(6), 1996, 1077-1085). 7. Thus, inhibition of nicotinic modulation in the central nervous system may result in inhibition of synaptic transmission and some of the behavioral consequences of general anesthesia. PMID:10049135

  7. Effect of homologous serotonin receptor loop substitutions on the heterologous expression in Pichia of a chimeric acetylcholine-binding protein with alpha-bungarotoxin-binding activity.

    PubMed

    Paulo, Joao A; Hawrot, Edward

    2009-10-01

    The molluscan acetylcholine-binding protein (AChBP) is a soluble homopentameric homolog of the extracellular domain of various ligand-gated ion channels. Previous studies have reported that AChBP, when fused to the ion pore domain of the serotonin receptor (5HT(3A)R), can form a functional ligand-gated chimeric channel only if the AChBP loop regions between beta-strands beta1 and beta2 (beta1-beta2), beta6 and beta7 (beta6-beta7), and beta8 and beta9 (beta8-beta9) are replaced with those of the 5HT(3A)R. To investigate further the potential interactions among these three important loop regions in a membrane- and detergent-free system, we designed AChBP constructs in which loops beta1-beta2, beta6-beta7, and beta8-beta9 of the AChBP were individually and combinatorially substituted in all permutations with the analogous loops of the 5HT(3A)R. These chimeras were expressed as secreted proteins using the Pichia pastoris yeast expression system. [(125)I]-alpha-Bungarotoxin-binding was detected in the culture media obtained from homologous recombinant clones expressing the wild-type AChBP, the beta1-beta2 loop-only chimera, and the chimera containing all three 5HT(3A)R loop substitutions. The remaining chimeras failed to show [(125)I]-alpha-bungarotoxin binding, and further analysis of cellular extracts allowed us to determine that these binding-negative chimeric constructs accumulated intracellularly and were not secreted into the culture medium. Our results demonstrate that coordinated interactions among loops beta1-beta2, beta6-beta7, and beta8-beta9 are essential for the formation of a functional ligand-binding site, as evidenced by [(125)I]-alpha-bungarotoxin-binding, and for efficient protein secretion. In addition, the constructs described here demonstrate the feasibility of utilizing soluble scaffolds to explore functionally important interactions within the extracellular domain of membrane-bound proteins. PMID:19427904

  8. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    PubMed Central

    Shah, Imran; Houck, Keith; Judson, Richard S.; Kavlock, Robert J.; Martin, Matthew T.; Reif, David M.; Wambaugh, John; Dix, David J.

    2011-01-01

    Background Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents. Results The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05). Conclusions The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the

  9. Xanthine oxidase, but not neutrophils, contributes to activation of cardiac sympathetic afferents during myocardial ischaemia in cats

    PubMed Central

    Tjen-A-Looi, Stephanie C; Fu, Liang-Wu; Longhurst, John C

    2002-01-01

    Activation of cardiac sympathetic afferents during myocardial ischaemia causes angina and induces important cardiovascular reflex responses. Reactive oxygen species (ROS) are important chemical stimuli of cardiac afferents during and after ischaemia. Iron-catalysed Fenton chemistry constitutes one mechanism of production of hydroxyl radicals. Another potential source of these species is xanthine oxidase-catalysed oxidation of purines. Polymorphonuclear leukocytes (PMNs) also contribute to the production of ROS in some conditions. The present study tested the hypothesis that both xanthine oxidase-catalysed oxidation of purines and neutrophils provide a source of ROS sufficient to activate cardiac afferents during ischaemia. We recorded single-unit activity of cardiac afferents innervating the ventricles recorded from the left thoracic sympathetic chain (T1-5) of anaesthetized cats to identify the afferents' responses to ischaemia. The role of xanthine oxidase in activation of these afferents was determined by infusion of oxypurinol (10 mg kg−1, i.v.), an inhibitor of xanthine oxidase. The importance of neutrophils as a potential source of ROS in the activation of cardiac afferents during ischaemia was assessed by the infusion of a polyclonal antibody (3 mg ml−1 kg−1, i.v.) raised in rabbits immunized with cat PMNs. This antibody decreased the number of circulating PMNs and, to a smaller extent, platelets. Since previous data suggest that platelets release serotonin (5-HT), which activates cardiac afferents through a serotonin receptor (subtype 3,5-HT3 receptor) mechanism, before treatment with the antibody in another group, we blocked 5-HT3 receptors on sensory nerve endings with tropisetron (300 μg kg−1, i.v.). We observed that oxypurinol significantly decreased the activity of cardiac afferents during myocardial ischaemia from 1.5 ± 0.4 to 0.8 ± 0.4 impulses s−1. Similarly, the polyclonal antibody significantly reduced the discharge frequency of

  10. NMDA Receptor Activity in Neuropsychiatric Disorders

    PubMed Central

    Lakhan, Shaheen E.; Caro, Mario; Hadzimichalis, Norell

    2013-01-01

    N-Methyl-d-aspartate (NMDA) receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington’s disease, Alzheimer’s disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms. PMID:23772215

  11. Mechanism of FGF receptor dimerization and activation

    NASA Astrophysics Data System (ADS)

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  12. Mechanism of FGF receptor dimerization and activation.

    PubMed

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise. PMID:26725515

  13. Mechanism of FGF receptor dimerization and activation

    PubMed Central

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise. PMID:26725515

  14. Characterization of the 5-hydroxytryptamine receptors mediating contraction in the pig isolated intravesical ureter

    PubMed Central

    Hernández, Medardo; Barahona, María Victoria; Simonsen, Ulf; Recio, Paz; Rivera, Luis; Martínez, Ana Cristina; García-Sacristán, Albino; Orensanz, Luis M; Prieto, Dolores

    2003-01-01

    This study was designed to investigate the effect of 5-hydroxytryptamine (5-HT) and to characterize the 5-HT receptors involved in 5-HT responses in the pig intravesical ureter. 5-HT (0.01–10 μM) concentration-dependently increased the tone of intravesical ureteral strips, whereas the increases in phasic contractions were concentration-independent. The 5-HT2 receptor agonist α-methyl 5-HT, mimicked the effect on tone whereas weak or no response was obtained with 5-CT, 8-OH-DPAT, m-chlorophenylbiguanide and RS 67333, 5-HT1, 5-HT1A, 5-HT3 and 5-HT4 receptor agonists, respectively. 5-HT did not induce relaxation of U46619-contracted ureteral preparations. Pargyline (100 μM), a monoaminooxidase A/B activity inhibitor, produced leftward displacements of the concentration-response curves for 5-HT. 5-HT-induced tone was reduced by the 5-HT2 and 5-HT2A receptor antagonists ritanserine (0.1 μM) and spiperone (0.2 μM), respectively. However, 5-HT contraction was not antagonized by cyanopindolol (2 μM), SDZ–SER 082 (1 μM), Y-25130 (1 μM) and GR 113808 (0.1 μM), which are respectively, 5-HT1A/1B, 5-HT2B/2C, 5-HT3, and 5-HT4 selective receptor antagonists. Removal of the urothelium did not modify 5-HT-induced contractions. Blockade of neuronal voltage-activated sodium channels, α-adrenergic receptors and adrenergic neurotransmission with tetrodotoxin (1 μM), phentolamine (0.3 μM) and guanethidine (10 μM), respectively, reduced the contractions to 5-HT. However, physostigmine (1 μM), atropine (0.1 μM) and suramin (30 μM), inhibitors of cholinesterase activity, muscarinic- and purinergic P2-receptors, respectively, failed to modify the contractions to 5-HT. These results suggest that 5-HT increases the tone of the pig intravesical ureter through 5-HT2A receptors located at the smooth muscle. Part of the 5-HT contraction is indirectly mediated via noradrenaline release from sympathetic nerves. PMID:12522083

  15. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed

    Jain, N; Kemp, N; Adeyemo, O; Buchanan, P; Stone, T W

    1995-10-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  16. Anxiolytic activity of adenosine receptor activation in mice.

    PubMed Central

    Jain, N.; Kemp, N.; Adeyemo, O.; Buchanan, P.; Stone, T. W.

    1995-01-01

    1. Purine analogues have been examined for anxiolytic- and anxiogenic-like activity in mice, by use of the elevated plus-maze. 2. The selective A1 receptor agonist, N6-cyclopentyladenosine (CPA) had marked anxiolytic-like activity at 10 and 50 microg kg(-1), with no effect on locomotor performance at these doses. 3. The A1 selective adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (CPX) had no significant effect on anxiety-related measures or locomotor behaviour, but blocked the anxiolytic-like activity of CPA. The hydrophilic xanthine, 8-(p-sulphophenyl) theophylline did not prevent anxiolysis by CPA. 4. Caffeine had anxiogenic-like activity at 30 mg kg(-1) which was prevented by CPA at 50 micro kg(-1). 5. The A2 receptor agonist, N6-[2-(3,5-dimethoxyphenyl)-2(2-methylphenyl)-ethyl]adenosine (DPMA) had no effect on anxiety behaviour but depressed locomotor activity at the highest dose tested of 1 mg kg(-1). The A2 receptor antagonist, 1,3-dimethyl-l-propargylxanthine (DMPX) had no effect on anxiety-related measures or locomotion and did not modify the anxiolytic-like activity of CPA. 6. Administration of DPMA in combination with anxiolytic doses of CPA prevented the anxiolytic-like activity of the latter. 7. The results suggest that the selective activation of central A1 adenosine receptors induces anxiolytic-like behaviour, while the activation of A2 sites causes locomotor depression and reduces the effects of A1 receptor activation. The absence of any effect of CPX alone suggests that the receptors involved in modulating behaviour in the elevated plus-maze in mice are not activated tonically by endogenous adenosine. PMID:8640355

  17. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  18. Synergistic effect of 5-hydroxytryptamine 3 and neurokinin 1 receptor antagonism in rodent models of somatic and visceral pain.

    PubMed

    Greenwood-Van Meerveld, Beverley; Mohammadi, Ehsan; Tyler, Karl; Pietra, Claudio; Bee, Lucy A; Dickenson, Anthony

    2014-10-01

    Synergistic activity has been observed between serotonergic 5-hydroxytryptamine 3 (5-HT3) and tachykinergic neurokinin 1 (NK1) receptor-mediated responses. This study investigated the efficacy of a 5-HT3 antagonist, palonosetron, and a NK1 antagonist, netupitant, alone or in combination in rodent models of somatic and visceral colonic hypersensitivity. In a rat model of experimental neuropathic pain, somatic hypersensitivity was quantified by the number of ipsilateral paw withdrawals to a von Frey filament (6g). Electrophysiologic responses were recorded in the dorsal horn neurons after mechanical or thermal stimuli. Acute colonic hypersensitivity was induced experimentally in rats by infusing dilute acetic acid (0.6%) directly into the colon. Colonic sensitivity was assessed by a visceromotor behavioral response quantified as the number of abdominal contractions in response to graded isobaric pressures (0-60 mm Hg) of colorectal distension. Palonosetron or netupitant was administered alone or in combination via oral gavage. When dosed alone, both significantly reduced somatic sensitivity, decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation, and caused significant (P < 0.05) inhibition of colonic hypersensitivity in a dose-dependent manner. The combined administration of palonosetron and netupitant at doses that were ineffective alone significantly reduced both somatic and visceral sensitivity and decreased the evoked response of spinal dorsal horn neurons to mechanical or thermal stimulation. In summary, the combination of palonosetron with a NK1 receptor antagonist showed synergistic analgesic activity in rodent models of somatic and visceral hypersensitivity, and may prove to be a useful therapeutic approach to treat pain associated with irritable bowel syndrome. PMID:25077526

  19. Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

    PubMed

    Minic, Zeljka; O'Leary, Donal S; Scislo, Tadeusz J

    2014-02-01

    Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8μg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension. PMID:24216055

  20. Progesterone receptors activation after acute cocaine administration.

    PubMed

    Wu, Hui-Bing K; Fabian, Sosimo; Jenab, Shirzad; Quiñones-Jenab, Vanya

    2006-12-18

    Cocaine modulates serum levels of progesterone in intact female and male rats, as well as in pregnant dams, and progesterone decreases or attenuates cocaine-induced behavioral and reward responses. It has been postulated that cocaine's modulation of serum progesterone levels may in turn alter progesterone receptor activity, thereby contributing to cocaine-induced alterations of neuronal functions and genomic regulations. To test this hypothesis, intact male rats received acute injections of saline or cocaine (15 or 30 mg/kg, dissolved in 0.9% saline, intraperitoneal). Progesterone serum levels, progesterone receptor (PR) protein levels, and PR-DNA binding complexes were measured in the striatum by radioimmunoassay, Western blot, and gel shift analyses, respectively. After injection of 15 mg/kg of cocaine, induction of progesterone serum levels was closely followed by an increase in receptor protein levels and DNA binding complexes. After injection of 30 mg/kg of cocaine, similar effects were observed along with an attenuation of receptor protein levels and DNA binding complexes at 60 min. Our results suggest that activation of progesterone receptors may be a mechanism by which cocaine mediates behavior through molecular alterations in the central nervous system. PMID:17109827

  1. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    EPA Science Inventory

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  2. Common mechanisms activate plant guard receptors and TLR4

    PubMed Central

    Kagan, Jonathan C.

    2014-01-01

    In metazoans, the innate immune system uses Pattern Recognition Receptors to detect conserved microbial products, whereas in plants Guard Receptors detect virulence factors or activities encoded by pathogens. In a recent study, Williams and colleagues report that plant Guard receptors can be activated by a mechanism remarkably similar to that of mammalian Toll-like Receptor 4. PMID:25224694

  3. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    PubMed

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  4. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor

    PubMed Central

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick

    2016-01-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)–forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally. PMID:26994072

  5. Activation Requirements for Metabotropic Glutamate Receptors

    PubMed Central

    Viaene, Angela N.; Petrof, Iraklis; Sherman, S. Murray

    2013-01-01

    It has been common experimentally to use high frequency, tetanic, stimulation to activate metabotropic glutamate receptors (mGluRs) in cortex and thalamus. To determine what type of stimulation is actually necessary to activate mGluRs we examined the effects of varying stimulation duration and intensity on activating mGluR responses. We used a thalamocortical and an intracortical slice preparation from mice and performed whole cell recordings from neurons in the ventral posterior medial nucleus or in layer 4 of primary somatosensory cortex (S1) while electrically stimulating in layer 6 of S1. Extracellular ionotropic glutamate receptor antagonists and GABAA receptor antagonists were used to isolate Group I or Group II mGluR responses. We observed that high frequency stimulation is not necessary for the activation of either Group I or Group II mGluRs. Either could be activated with as few as 2-3 pulses at stimulation frequencies around 15-20Hz. Additionally, increasing the number of pulses, intensity of stimulation, or stimulation frequency increased amplitude and duration of the mGluR response. PMID:23416319

  6. Equivalent Activities of Repulsive Axon Guidance Receptors

    PubMed Central

    Long, Hong; Yoshikawa, Shingo

    2016-01-01

    Receptors on the growth cone at the leading edge of elongating axons play critical guidance roles by recognizing cues via their extracellular domains and transducing signals via their intracellular domains, resulting in changes in direction of growth. An important concept to have emerged in the axon guidance field is the importance of repulsion as a major guidance mechanism. Given the number and variety of different repulsive receptors, it is generally thought that there are likely to be qualitative differences in the signals they transduce. However, the nature of these possible differences is unknown. By creating chimeras using the extracellular and intracellular domains of three different Drosophila repulsive receptors, Unc5, Roundabout (Robo), and Derailed (Drl) and expressing them in defined cells within the embryonic nervous system, we examined the responses elicited by their intracellular domains systematically. Surprisingly, we found no qualitative differences in growth cone response or axon growth, suggesting that, despite their highly diverged sequences, each intracellular domain elicits repulsion via a common pathway. In terms of the signaling pathway(s) used by the repulsive receptors, mutations in the guanine nucleotide exchange factor Trio strongly enhance the repulsive activity of all three intracellular domains, suggesting that repulsion by Unc5, Robo, and Drl, and perhaps repulsion in general, involves Trio activity. SIGNIFICANCE STATEMENT A prevailing concept that has emerged in the axon guidance field is the importance of repulsion as a guidance mechanism for steering axons to their appropriate targets. Given the number and variety of different repulsive receptors, it is generally thought that there are differences in the signals that they transduce. However, this has never been tested directly. We have used the advanced genetics of Drosophila to compare directly the outputs of different repulsive receptors. Surprisingly, we found no qualitative

  7. Structural requirements of bitter taste receptor activation

    PubMed Central

    Brockhoff, Anne; Behrens, Maik; Niv, Masha Y.; Meyerhof, Wolfgang

    2010-01-01

    An important question in taste research is how 25 receptors of the human TAS2R family detect thousands of structurally diverse compounds. An answer to this question may arise from the observation that TAS2Rs in general are broadly tuned to interact with numerous substances. Ultimately, interaction with chemically diverse agonists requires architectures of binding pockets tailored to combine flexibility with selectivity. The present study determines the structure of hTAS2R binding pockets. We focused on a subfamily of closely related hTAS2Rs exhibiting pronounced amino acid sequence identities but unique agonist activation spectra. The generation of chimeric and mutant receptors followed by calcium imaging analyses identified receptor regions and amino acid residues critical for activation of hTAS2R46, -R43, and -R31. We found that the carboxyl-terminal regions of the investigated receptors are crucial for agonist selectivity. Intriguingly, exchanging two residues located in transmembrane domain seven between hTAS2R46, activated by strychnine, and hTAS2R31, activated by aristolochic acid, was sufficient to invert agonist selectivity. Further mutagenesis revealed additional positions involved in agonist interaction. The transfer of functionally relevant amino acids identified in hTAS2R46 to the corresponding positions of hTAS2R43 and -R31 resulted in pharmacological properties indistinguishable from the parental hTAS2R46. In silico modeling of hTAS2R46 allowed us to visualize the putative mode of interaction between agonists and hTAS2Rs. Detailed structure-function analyses of hTAS2Rs may ultimately pave the way for the development of specific antagonists urgently needed for more sophisticated analyses of human bitter taste perception. PMID:20534469

  8. Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

    PubMed Central

    2013-01-01

    Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

  9. Unbalanced Peptidergic Inhibition in Superficial Neocortex Underlies Spike and Wave Seizure Activity

    PubMed Central

    Hall, S.; Hunt, M.; Simon, A.; Cunnington, L.G.; Carracedo, L.M.; Schofield, I.S.; Forsyth, R.; Traub, R.D.

    2015-01-01

    Slow spike and wave discharges (0.5–4 Hz) are a feature of many epilepsies. They are linked to pathology of the thalamocortical axis and a thalamic mechanism has been elegantly described. Here we present evidence for a separate generator in local circuits of associational areas of neocortex manifest from a background, sleep-associated delta rhythm in rat. Loss of tonic neuromodulatory excitation, mediated by nicotinic acetylcholine or serotonin (5HT3A) receptors, of 5HT3-immunopositive interneurons caused an increase in amplitude and slowing of the delta rhythm until each period became the “wave” component of the spike and wave discharge. As with the normal delta rhythm, the wave of a spike and wave discharge originated in cortical layer 5. In contrast, the “spike” component of the spike and wave discharge originated from a relative failure of fast inhibition in layers 2/3—switching pyramidal cell action potential outputs from single, sparse spiking during delta rhythms to brief, intense burst spiking, phase-locked to the field spike. The mechanisms underlying this loss of superficial layer fast inhibition, and a concomitant increase in slow inhibition, appeared to be precipitated by a loss of neuropeptide Y (NPY)-mediated local circuit inhibition and a subsequent increase in vasoactive intestinal peptide (VIP)-mediated disinhibition. Blockade of NPY Y1 receptors was sufficient to generate spike and wave discharges, whereas blockade of VIP receptors almost completely abolished this form of epileptiform activity. These data suggest that aberrant, activity-dependent neuropeptide corelease can have catastrophic effects on neocortical dynamics. PMID:26109655

  10. Origin of basal activity in mammalian olfactory receptor neurons

    PubMed Central

    2010-01-01

    Mammalian odorant receptors form a large, diverse group of G protein–coupled receptors that determine the sensitivity and response profile of olfactory receptor neurons. But little is known if odorant receptors control basal and also stimulus-induced cellular properties of olfactory receptor neurons other than ligand specificity. This study demonstrates that different odorant receptors have varying degrees of basal activity, which drives concomitant receptor current fluctuations and basal action potential firing. This basal activity can be suppressed by odorants functioning as inverse agonists. Furthermore, odorant-stimulated olfactory receptor neurons expressing different odorant receptors can have strikingly different response patterns in the later phases of prolonged stimulation. Thus, the influence of odorant receptor choice on response characteristics is much more complex than previously thought, which has important consequences on odor coding and odor information transfer to the brain. PMID:20974772

  11. Fatty acid activation of peroxisome proliferator-activated receptor (PPAR).

    PubMed

    Bocos, C; Göttlicher, M; Gearing, K; Banner, C; Enmark, E; Teboul, M; Crickmore, A; Gustafsson, J A

    1995-06-01

    Peroxisome proliferators such as clofibric acid, nafenopin, and WY-14,643 have been shown to activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily. We have cloned the cDNA from rat that is homologous to that from mouse, which encodes a 97% similar protein. To search for physiologically occurring activators, we established a transcriptional transactivation assay by stably expressing in CHO cells a chimera of rat PPAR and the human glucocorticoid receptor that activates expression of the placental alkaline phosphatase reporter gene under the control of the mouse mammary tumor virus promoter. 150 microM concentrations of arachidonic or linoleic acid but not of dehydroepiandrosterone, cholesterol, or 25-hydroxy-cholesterol, activated the receptor chimera. In addition, saturated fatty acids induced the reporter gene. Shortening the chain length to n = 6 or introduction of an omega-terminal carboxylic group abolished the activation potential of the fatty acid. To test whether a common PPAR binding metabolite might be formed from free fatty acids we tested the effects of differentially beta-oxidizable fatty acids and inhibitors of fatty acid metabolism. The peroxisomal proliferation-inducing, non-beta-oxidizable, tetradecylthioacetic acid activated PPAR to the same extent as the strong peroxisomal proliferator WY-14,643, whereas the homologous beta-oxidizable tetradecylthiopropionic acid was only as potent as a non-substituted fatty acid. Cyclooxygenase inhibitors, radical scavengers or cytochrome P450 inhibitors did not affect activation of PPAR. In conclusion, beta-oxidation is apparently not required for the formation of the PPAR-activating molecule and this moiety might be a fatty acid, its ester with CoA, or a further derivative of the activated fatty acid prior to beta-oxidation of the acyl-CoA ester. PMID:7626496

  12. Monoclonal Antibodies to the Human Insulin Receptor that Activate Glucose Transport but not Insulin Receptor Kinase Activity

    NASA Astrophysics Data System (ADS)

    Forsayeth, John R.; Caro, Jose F.; Sinha, Madhur K.; Maddux, Betty A.; Goldfine, Ira D.

    1987-05-01

    Three mouse monoclonal antibodies were produced that reacted with the α subunit of the human insulin receptor. All three both immunoprecipitated 125I-labeled insulin receptors from IM-9 lymphocytes and competitively inhibited 125I-labeled insulin binding to its receptor. Unlike insulin, the antibodies failed to stimulate receptor autophosphorylation in both intact IM-9 lymphocytes and purified human placental insulin receptors. Moreover, unlike insulin, the antibodies failed to stimulate receptor-mediated phosphorylation of exogenous substrates. However, like insulin, two of the three antibodies stimulated glucose transport in isolated human adipocytes. One antibody, on a molar basis, was as potent as insulin. These studies indicate, therefore, that monoclonal antibodies to the insulin receptor can mimic a major function of insulin without activating receptor kinase activity. They also raise the possibility that certain actions of insulin such as stimulation of glucose transport may not require the activation of receptor kinase activity.

  13. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    PubMed Central

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra; Roncaglioni, Alessandra; Tropsha, Alexander; Varnek, Alexandre; Zakharov, Alexey; Worth, Andrew; Richard, Ann M.; Grulke, Christopher M.; Trisciuzzi, Daniela; Fourches, Denis; Horvath, Dragos; Benfenati, Emilio; Muratov, Eugene; Wedebye, Eva Bay; Grisoni, Francesca; Mangiatordi, Giuseppe F.; Incisivo, Giuseppina M.; Hong, Huixiao; Ng, Hui W.; Tetko, Igor V.; Balabin, Ilya; Kancherla, Jayaram; Shen, Jie; Burton, Julien; Nicklaus, Marc; Cassotti, Matteo; Nikolov, Nikolai G.; Nicolotti, Orazio; Andersson, Patrik L.; Zang, Qingda; Politi, Regina; Beger, Richard D.; Todeschini, Roberto; Huang, Ruili; Farag, Sherif; Rosenberg, Sine A.; Slavov, Svetoslav; Hu, Xin; Judson, Richard S.

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. Objectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) and demonstrate the efficacy of using predictive computational models trained on high-throughput screening data to evaluate thousands of chemicals for ER-related activity and prioritize them for further testing. Methods: CERAPP combined multiple models developed in collaboration with 17 groups in the United States and Europe to predict ER activity of a common set of 32,464 chemical structures. Quantitative structure–activity relationship models and docking approaches were employed, mostly using a common training set of 1,677 chemical structures provided by the U.S. EPA, to build a total of 40 categorical and 8 continuous models for binding, agonist, and antagonist ER activity. All predictions were evaluated on a set of 7,522 chemicals curated from the literature. To overcome the limitations of single models, a consensus was built by weighting models on scores based on their evaluated accuracies. Results: Individual model scores ranged from 0.69 to 0.85, showing high prediction reliabilities. Out of the 32,464 chemicals, the consensus model predicted 4,001 chemicals (12.3%) as high priority actives and 6,742 potential actives (20.8%) to be considered for further testing. Conclusion: This project demonstrated the possibility to screen large libraries of chemicals using a consensus of different in silico approaches. This concept will be applied in future projects related to other

  14. How IGF-1 activates its receptor

    PubMed Central

    Kavran, Jennifer M; McCabe, Jacqueline M; Byrne, Patrick O; Connacher, Mary Katherine; Wang, Zhihong; Ramek, Alexander; Sarabipour, Sarvenaz; Shan, Yibing; Shaw, David E; Hristova, Kalina; Cole, Philip A; Leahy, Daniel J

    2014-01-01

    The type I insulin-like growth factor receptor (IGF1R) is involved in growth and survival of normal and neoplastic cells. A ligand-dependent conformational change is thought to regulate IGF1R activity, but the nature of this change is unclear. We point out an underappreciated dimer in the crystal structure of the related Insulin Receptor (IR) with Insulin bound that allows direct comparison with unliganded IR and suggests a mechanism by which ligand regulates IR/IGF1R activity. We test this mechanism in a series of biochemical and biophysical assays and find the IGF1R ectodomain maintains an autoinhibited state in which the TMs are held apart. Ligand binding releases this constraint, allowing TM association and unleashing an intrinsic propensity of the intracellular regions to autophosphorylate. Enzymatic studies of full-length and kinase-containing fragments show phosphorylated IGF1R is fully active independent of ligand and the extracellular-TM regions. The key step triggered by ligand binding is thus autophosphorylation. DOI: http://dx.doi.org/10.7554/eLife.03772.001 PMID:25255214

  15. Serotonin induces the migration of PC12 cells via the serotonin receptor 6/cAMP/ERK pathway

    PubMed Central

    KOIZUMI, KEITA; NAKAJIMA, HIDEO

    2014-01-01

    Serotonin (5-HT) functions as a chemoattractant that modulates neural migration during prenatal and early postnatal development. However, its molecular mechanism remains to be elucidated. The effect of 5-HT on neural cell migration was examined using PC12 neuron-like cell line. Transwell migration assay was used to determine the effect of 5-HT on PC12 cell migration. The results demonstrated that 5-HT and nerve growth factor (NGF) induced PC12 cell migration in a dose-dependent manner. Additionally, 5-HT receptor antagonists suggest that 5-HT-induced migration was mediated by serotonin receptor 6 (5-HT6), a Gs-protein coupled receptor that elevates the intercellular cAMP level. By contrast, antagonists of serotonin receptor 3 (5-HT3) did not show any effects on PC12 cell migration. Clozapine, an inhibitor of cAMP accumulation mediated by 5-HT6, significantly reduced the effect of 5-HT on the PC12 cell migration. An inhibitor of extracellular signal-regulated kinase (ERK) also suppressed migration. These results suggest that 5-HT induces PC12 cell migration by activating cAMP/ERK signaling pathways, which is mediated by 5-HT6 receptor. PMID:24649064

  16. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    EPA Science Inventory

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  17. Dendritic NMDA receptors activate axonal calcium channels

    PubMed Central

    Christie, Jason M.; Jahr, Craig E.

    2008-01-01

    Summary NMDA receptor (NMDAR) activation can alter synaptic strength by regulating transmitter release from a variety of neurons in the CNS. As NMDARs are permeable to Ca2+ and monovalent cations, they could alter release directly by increasing presynaptic Ca2+ or indirectly by axonal depolarization sufficient to activate voltage-sensitive Ca2+ channels (VSCCs). Using two-photon microscopy to measure Ca2+ excursions, we found that somatic depolarization or focal activation of dendritic NMDARs elicited small Ca2+ transients in axon varicosities of cerebellar stellate cell interneurons. These axonal transients resulted from Ca2+ entry through VSCCs that were opened by the electrotonic spread of the NMDAR-mediated depolarization elicited in the dendrites. In contrast, we were unable to detect direct activation of NMDARs on axons indicating an exclusive somatodendritic expression of functional NMDARs. In cerebellar stellate cells, dendritic NMDAR activation masquerades as a presynaptic phenomenon and may influence Ca2+-dependent forms of presynaptic plasticity and release. PMID:18957221

  18. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    SciTech Connect

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J.

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  19. Activation of neurotensin receptor type 1 attenuates locomotor activity.

    PubMed

    Vadnie, Chelsea A; Hinton, David J; Choi, Sun; Choi, YuBin; Ruby, Christina L; Oliveros, Alfredo; Prieto, Miguel L; Park, Jun Hyun; Choi, Doo-Sup

    2014-10-01

    Intracerebroventricular administration of neurotensin (NT) suppresses locomotor activity. However, the brain regions that mediate the locomotor depressant effect of NT and receptor subtype-specific mechanisms involved are unclear. Using a brain-penetrating, selective NT receptor type 1 (NTS1) agonist PD149163, we investigated the effect of systemic and brain region-specific NTS1 activation on locomotor activity. Systemic administration of PD149163 attenuated the locomotor activity of C57BL/6J mice both in a novel environment and in their homecage. However, mice developed tolerance to the hypolocomotor effect of PD149163 (0.1 mg/kg, i.p.). Since NTS1 is known to modulate dopaminergic signaling, we examined whether PD149163 blocks dopamine receptor-mediated hyperactivity. Pretreatment with PD149163 (0.1 or 0.05 mg/kg, i.p.) inhibited D2R agonist bromocriptine (8 mg/kg, i.p.)-mediated hyperactivity. D1R agonist SKF-81297 (8 mg/kg, i.p.)-induced hyperlocomotion was only inhibited by 0.1 mg/kg of PD149163. Since the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) have been implicated in the behavioral effects of NT, we examined whether microinjection of PD149163 into these regions reduces locomotion. Microinjection of PD149163 (2 pmol) into the NAc, but not the mPFC suppressed locomotor activity. In summary, our results indicate that systemic and intra-NAc activation of NTS1 is sufficient to reduce locomotion and NTS1 activation inhibits D2R-mediated hyperactivity. Our study will be helpful to identify pharmacological factors and a possible therapeutic window for NTS1-targeted therapies for movement disorders. PMID:24929110

  20. Aryl biphenyl-3-ylmethylpiperazines as 5-HT7 receptor antagonists.

    PubMed

    Kim, Jeeyeon; Kim, Youngjae; Tae, Jinsung; Yeom, Miyoung; Moon, Bongjin; Huang, Xi-Ping; Roth, Bryan L; Lee, Kangho; Rhim, Hyewhon; Choo, Il Han; Chong, Youhoon; Keum, Gyochang; Nam, Ghilsoo; Choo, Hyunah

    2013-11-01

    The 5-HT7 receptor (5-HT7 R) is a promising therapeutic target for the treatment of depression and neuropathic pain. The 5-HT7 R antagonist SB-269970 exhibited antidepressant-like activity, whereas systemic administration of the 5-HT7 R agonist AS-19 significantly inhibited mechanical hypersensitivity and thermal hyperalgesia. In our efforts to discover selective 5-HT7 R antagonists or agonists, aryl biphenyl-3-ylmethylpiperazines were designed, synthesized, and biologically evaluated against the 5-HT7 R. Among the synthesized compounds, 1-([2'-methoxy-(1,1'-biphenyl)-3-yl]methyl)-4-(2-methoxyphenyl)piperazine (28) was the best binder to the 5-HT7 R (pKi =7.83), and its antagonistic property was confirmed by functional assays. The selectivity profile of compound 28 was also recorded for the 5-HT7 R over other serotonin receptor subtypes, such as 5-HT1 R, 5-HT2 R, 5-HT3 R, and 5-HT6 R. In a molecular modeling study, the 2-methoxyphenyl moiety attached to the piperazine ring of compound 28 was proposed to be essential for the antagonistic function. PMID:24039134

  1. Prophylaxis of Radiation-Induced Nausea and Vomiting Using 5-Hydroxytryptamine-3 Serotonin Receptor Antagonists: A Systematic Review of Randomized Trials

    SciTech Connect

    Salvo, Nadia; Doble, Brett; Khan, Luluel; Amirthevasar, Gayathri; Dennis, Kristopher; Pasetka, Mark; DeAngelis, Carlo; Tsao, May; Chow, Edward

    2012-01-01

    Purpose: To systematically review the effectiveness and safety of 5-hydroxytryptamine-3 receptor antagonists (5-HT3 RAs) compared with other antiemetic medication or placebo for prophylaxis of radiation-induced nausea and vomiting. Methods and Materials: We searched the following electronic databases: MEDLINE, Embase, the Cochrane Central Register of Controlled Clinical Trials, and Web of Science. We also hand-searched reference lists of included studies. Randomized, controlled trials that compared a 5-HT3 RA with another antiemetic medication or placebo for preventing radiation-induced nausea and vomiting were included. We excluded studies recruiting patients receiving concomitant chemotherapy. When appropriate, meta-analysis was conducted using Review Manager (v5) software. Relative risks were calculated using inverse variance as the statistical method under a random-effects model. We assessed the quality of evidence by outcome using the Grading of Recommendations Assessment, Development, and Evaluation approach. Results: Eligibility screening of 47 articles resulted in 9 included in the review. The overall methodologic quality was moderate. Meta-analysis of 5-HT3 RAs vs. placebo showed significant benefit for 5-HT3 RAs (relative risk [RR] 0.70; 95% confidence interval [CI] 0.57-0.86 for emesis; RR 0.84, 95% CI 0.73-0.96 for nausea). Meta-analysis comparing 5-HT3 RAs vs. metoclopramide showed a significant benefit of the 5-HT3 RAs for emetic control (RR 0.27, 95% CI 0.15-0.47). Conclusion: 5-Hydroxytryptamine-3 RAs are superior to placebo and other antiemetics for prevention of emesis, but little benefit was identified for nausea prevention. 5-Hydroxytryptamine-3 RAs are suggested for prevention of emesis. Limited evidence was found regarding delayed emesis, adverse events, quality of life, or need for rescue medication. Future randomized, controlled trials should evaluate different 5-HT3 antiemetics and new agents with novel mechanisms of action such at the NK

  2. Involvement of 5-HT₇ receptors in vortioxetine's modulation of circadian rhythms and episodic memory in rodents.

    PubMed

    Westrich, Ligia; Haddjeri, Nasser; Dkhissi-Benyahya, Ouria; Sánchez, Connie

    2015-02-01

    Since poor circadian synchrony and cognitive dysfunction have been linked to affective disorders, antidepressants that target key 5-HT (serotonin) receptor subtypes involved in circadian rhythm and cognitive regulation may have therapeutic utility. Vortioxetine is a multimodal antidepressant that inhibits 5-HT1D, 5-HT3, 5-HT7 receptor activity, 5-HT reuptake, and enhances the activity of 5-HT1A and 5-HT1B receptors. In this study, we investigated the effects of vortioxetine on the period length of PER2::LUC expression, circadian behavior, and episodic memory, using tissue explants from genetically modified PER2::LUC mice, locomotor activity rhythm monitoring, and the object recognition test, respectively. Incubation of tissue explants from the suprachiasmatic nucleus of PER2::LUC mice with 0.1 μM vortioxetine increased the period length of PER2 bioluminescence. Monitoring of daily wheel-running activity of Sprague-Dawley rats treated with vortioxetine (10 mg/kg, s.c.), alone or in combination with the 5-HT1A receptor agonist flesinoxan (2.5 mg/kg, s.c.) or the 5-HT7 receptor antagonist SB269970 (30 mg/kg, s.c.), just prior to activity onset revealed significant delays in wheel-running behavior. The increase in circadian period length and the phase delay produced by vortioxetine were abolished in the presence of the 5-HT7 receptor partial agonist AS19. Finally, in the object recognition test, vortioxetine (10 mg/kg, i.p.) increased the time spent exploring the novel object during the retention test and this effect was prevented by AS19 (5 mg/kg, i.p.). In conclusion, the present study shows that vortioxetine, partly via its 5-HT7 receptor antagonism, induced a significant effect on circadian rhythm and presented promnesic properties in rodents. PMID:25446573

  3. Composition, assembly and activation of the avian progesterone receptor.

    PubMed

    Smith, D F; Toft, D O

    1992-03-01

    When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30 degrees C), (3) presence of ATP, and (4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system. PMID:1562503

  4. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties.

    PubMed

    Watkins, Harriet A; Chakravarthy, Madhuri; Abhayawardana, Rekhati S; Gingell, Joseph J; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M W R; Lathbridge, Alex; Constantine, Arran; Harris, Paul W R; Yuen, Tsz-Ying; Brimble, Margaret A; Barwell, James; Poyner, David R; Woolley, Michael J; Conner, Alex C; Pioszak, Augen A; Reynolds, Christopher A; Hay, Debbie L

    2016-05-27

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  5. Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties*

    PubMed Central

    Watkins, Harriet A.; Chakravarthy, Madhuri; Abhayawardana, Rekhati S.; Gingell, Joseph J.; Garelja, Michael; Pardamwar, Meenakshi; McElhinney, James M. W. R.; Lathbridge, Alex; Constantine, Arran; Harris, Paul W. R.; Yuen, Tsz-Ying; Brimble, Margaret A.; Barwell, James; Poyner, David R.; Woolley, Michael J.; Conner, Alex C.; Pioszak, Augen A.; Reynolds, Christopher A.

    2016-01-01

    Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM1 and AM2 receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM1 and AM2 receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM1 and AM2 receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function. PMID:27013657

  6. The insulin receptor C-terminus is involved in regulation of the receptor kinase activity.

    PubMed

    Kaliman, P; Baron, V; Alengrin, F; Takata, Y; Webster, N J; Olefsky, J M; Van Obberghen, E

    1993-09-21

    During the insulin receptor activation process, ligand binding and autophosphorylation induce two distinct conformational changes in the C-terminal domain of the receptor beta-subunit. To analyze the role of this domain and the involvement of the C-terminal autophosphorylation sites (Tyr1316 and Tyr1322) in receptor activation, we used (i) antipeptide antibodies against three different C-terminal sequences (1270-1281, 1294-1317, and 1309-1326) and (ii) an insulin receptor mutant (Y/F2) where Tyr1316 and Tyr1322 have been replaced by Phe. We show that the autophosphorylation-induced C-terminal conformational change is preserved in the Y/F2 receptor, indicating that this change is not induced by phosphorylation of the C-terminal sites but most likely by phosphorylation of the major sites in the kinase domain (Tyr1146, Tyr1150, and Tyr1151). Binding of antipeptide antibodies to the C-terminal domain modulated (activated or inhibited) both mutant and wild-type receptor-mediated phosphorylation of poly(Glu/Tyr). In contrast to the wild-type receptor, Y/F2 exhibited the same C-terminal configuration before and after insulin binding, evidencing that mutation of Tyr1316 and Tyr1322 introduced conformational changes in the C-terminus. Finally, the mutant receptor was 2-fold more active than the wild-type receptor for poly(Glu/Tyr) phosphorylation. In conclusion, the whole C-terminal region of the insulin receptor beta-subunit is likely to exert a regulatory influence on the receptor kinase activity. Perturbations of the C-terminal region, such as binding of antipeptides or mutation of Tyr1316 and Tyr1322, provoke alterations at the receptor kinase level, leading to activation or inhibition of the enzymic activity. PMID:7690586

  7. Constitutive Activity of the Androgen Receptor

    PubMed Central

    Chan, Siu Chiu; Dehm, Scott M.

    2014-01-01

    Prostate cancer (PCa) is the most frequently diagnosed cancer in the United States. The androgen receptor (AR) signaling axis is central to all stages of PCa pathophysiology and serves as the main target for endocrine-based therapy. The most advanced stage of the disease, castration resistant prostate cancer (CRPC), is presently incurable and accounts for most PCa mortality. In this review, we highlight the mechanisms by which the AR signaling axis can bypass endocrine-targeted therapies and drive progression of CRPC. These mechanisms include alterations in growth factor, cytokine, and inflammatory signaling pathways, altered expression or activity of transcriptional co-regulators, AR point mutations, and AR gene amplification leading to AR protein overexpression. Additionally, we will discuss the mechanisms underlying the synthesis of constitutively active AR splice variants (AR-Vs) lacking the COOH-terminal ligand binding domain, as well as the role and regulation of AR-Vs in supporting therapeutic resistance in CRPC. Finally, we summarize the ongoing development of inhibitors targeting discrete AR functional domains as well as the status of new biomarkers for monitoring the AR signaling axis in patients. PMID:24931201

  8. Thyroid hormone receptors regulate adipogenesis and carcinogenesis via crosstalk signaling with peroxisome proliferator-activated receptors

    PubMed Central

    Lu, Changxue; Cheng, Sheue-Yann

    2012-01-01

    Peroxisome proliferator-activated receptors (PPARs) and thyroid hormone receptors (TRs) are members of the nuclear receptor superfamily. They are ligand-dependent transcription factors that interact with their cognate hormone response elements in the promoters to regulate respective target gene expression to modulate cellular functions. While the transcription activity of each is regulated by their respective ligands, recent studies indicate that via multiple mechanisms PPARs and TRs crosstalk to affect diverse biological functions. Here, we review recent advances in the understanding of the molecular mechanisms and biological impact of crosstalk between these two important nuclear receptors, focusing on their roles in adipogenesis and carcinogenesis. PMID:19741045

  9. Mechanisms of xenobiotic receptor activation: Direct vs. indirect.

    PubMed

    Mackowiak, Bryan; Wang, Hongbing

    2016-09-01

    The so-called xenobiotic receptors (XRs) have functionally evolved into cellular sensors for both endogenous and exogenous stimuli by regulating the transcription of genes encoding drug-metabolizing enzymes and transporters, as well as those involving energy homeostasis, cell proliferation, and/or immune responses. Unlike prototypical steroid hormone receptors, XRs are activated through both direct ligand-binding and ligand-independent (indirect) mechanisms by a plethora of structurally unrelated chemicals. This review covers research literature that discusses direct vs. indirect activation of XRs. A particular focus is centered on the signaling control of the constitutive androstane receptor (CAR), the pregnane X receptor (PXR), and the aryl hydrocarbon receptor (AhR). We expect that this review will shed light on both the common and distinct mechanisms associated with activation of these three XRs. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie. PMID:26877237

  10. Cell death sensitization of leukemia cells by opioid receptor activation

    PubMed Central

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  11. Comparison of the activation kinetics of the M3 acetylcholine receptor and a constitutively active mutant receptor in living cells.

    PubMed

    Hoffmann, Carsten; Nuber, Susanne; Zabel, Ulrike; Ziegler, Nicole; Winkler, Christiane; Hein, Peter; Berlot, Catherine H; Bünemann, Moritz; Lohse, Martin J

    2012-08-01

    Activation of G-protein-coupled receptors is the first step of the signaling cascade triggered by binding of an agonist. Here we compare the activation kinetics of the G(q)-coupled M(3) acetylcholine receptor (M(3)-AChR) with that of a constitutively active mutant receptor (M(3)-AChR-N514Y) using M(3)-AChR constructs that report receptor activation by changes in the fluorescence resonance energy transfer (FRET) signal. We observed a leftward shift in the concentration-dependent FRET response for acetylcholine and carbachol with M(3)-AChR-N514Y. Consistent with this result, at submaximal agonist concentrations, the activation kinetics of M(3)-AChR-N514Y were significantly faster, whereas at maximal agonist concentrations the kinetics of receptor activation were identical. Receptor deactivation was significantly faster with carbachol than with acetylcholine and was significantly delayed by the N514Y mutation. Receptor-G-protein interaction was measured by FRET between M(3)-AChR-yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP)-Gγ(2). Agonist-induced receptor-G-protein coupling was of a time scale similar to that of receptor activation. As observed for receptor deactivation, receptor-G-protein dissociation was slower for acetylcholine than that for carbachol. Acetylcholine-stimulated increases in receptor-G-protein coupling of M(3)-AChR-N514Y reached only 12% of that of M(3)-AChR and thus cannot be kinetically analyzed. G-protein activation was measured using YFP-tagged Gα(q) and CFP-tagged Gγ(2). Activation of G(q) was significantly slower than receptor activation and indistinguishable for the two agonists. However, G(q) deactivation was significantly prolonged for acetylcholine compared with that for carbachol. Consistent with decreased agonist-stimulated coupling to G(q), agonist-stimulated G(q) activation by M(3)-AChR-N514Y was not detected. Taken together, these results indicate that the N514Y mutation produces constitutive activation of M(3

  12. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    SciTech Connect

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  13. The insulin receptor activation process involves localized conformational changes.

    PubMed

    Baron, V; Kaliman, P; Gautier, N; Van Obberghen, E

    1992-11-15

    The molecular process by which insulin binding to the receptor alpha-subunit induces activation of the receptor beta-subunit with ensuing substrate phosphorylation remains unclear. In this study, we aimed at approaching this molecular mechanism of signal transduction and at delineating the cytoplasmic domains implied in this process. To do this, we used antipeptide antibodies to the following sequences of the receptor beta-subunit: (i) positions 962-972 in the juxtamembrane domain, (ii) positions 1247-1261 at the end of the kinase domain, and (iii) positions 1294-1317 and (iv) positions 1309-1326, both in the receptor C terminus. We have previously shown that insulin binding to its receptor induces a conformational change in the beta-subunit C terminus. Here, we demonstrate that receptor autophosphorylation induces an additional conformational change. This process appears to be distinct from the one produced by ligand binding and can be detected in at least three different beta-subunit regions: the juxtamembrane domain, the kinase domain, and the C terminus. Hence, the cytoplasmic part of the receptor beta-subunit appears to undergo an extended conformational change upon autophosphorylation. By contrast, the insulin-induced change does not affect the juxtamembrane domain 962-972 nor the kinase domain 1247-1261 and may be limited to the receptor C terminus. Further, we show that the hormone-dependent conformational change is maintained in a kinase-deficient receptor due to a mutation at lysine 1018. Therefore, during receptor activation, the ligand-induced change could precede ATP binding and receptor autophosphorylation. We propose that insulin binding leads to a transient receptor form that may allow ATP binding and, subsequently, autophosphorylation. The second conformational change could unmask substrate-binding sites and stabilize the receptor in an active conformation. PMID:1331080

  14. Steroid receptor RNA activator: Biologic function and role in disease.

    PubMed

    Liu, Chan; Wu, Hong-Tao; Zhu, Neng; Shi, Ya-Ning; Liu, Zheng; Ao, Bao-Xue; Liao, Duan-Fang; Zheng, Xi-Long; Qin, Li

    2016-08-01

    Steroid receptor RNA activator (SRA) is a type of long noncoding RNA (lncRNA) which coordinates the functions of various transcription factors, enhances steroid receptor-dependent gene expression, and also serves as a distinct scaffold. The novel, profound and expanded roles of SRA are emerging in critical aspects of coactivation of nuclear receptors (NRs). As a nuclear receptor coactivator, SRA can coactivate androgen receptor (AR), estrogen receptor α (ERα), ERβ, progesterone receptor (PR), glucocorticoid receptor (GR), thyroid hormone receptor and retinoic acid receptor (RAR). Although SRA is one of the least well-understood molecules, increasing studies have revealed that SRA plays a key role in both biological processes, such as myogenesis and steroidogenesis, and pathological changes, including obesity, cardiomyopathy, and tumorigenesis. Furthermore, the SRA-related signaling pathways, such as the mitogen-activated protein kinase (p38 MAPK), Notch and tumor necrosis factor α (TNFα) pathways, play critical roles in the pathogenesis of estrogen-dependent breast cancers. In addition, the most recent data demonstrates that SRA expression may serve as a new prognostic marker in patients with ER-positive breast cancer. Thus, elucidating the molecular mechanisms underlying SRA-mediated functions is important to develop proper novel strategies to target SRA in the diagnosis and treatment of human diseases. PMID:27282881

  15. The novel platelet activation receptor CLEC-2.

    PubMed

    Suzuki-Inoue, Katsue; Inoue, Osamu; Ozaki, Yukio

    2011-01-01

    The c-type lectin-like receptor 2 (CLEC-2) was first identified from a bio-informatic screen for c-type lectin-like receptors. However, neither its function nor its ligand(s) had been elucidated for several years. In 2006, we reported that the receptor is expressed on the surface of platelets and serves as a receptor for the snake venom rhodocytin, which potently stimulates platelet aggregation. Since then CLEC-2 has been intensively investigated, and its endogenous/exogenous ligands and several physiological/pathological roles have been clarified. In this article and its accompanying poster, we outline the structure, distribution, signal transduction mechanism and functions of CLEC-2. PMID:21714702

  16. Interdicting Gq Activation in Airway Disease by Receptor-Dependent and Receptor-Independent Mechanisms.

    PubMed

    Carr, Richard; Koziol-White, Cynthia; Zhang, Jie; Lam, Hong; An, Steven S; Tall, Gregory G; Panettieri, Reynold A; Benovic, Jeffrey L

    2016-01-01

    Gαqβγ heterotrimer (Gq), an important mediator in the pathology of airway disease, plays a central role in bronchoconstriction and airway remodeling, including airway smooth muscle growth and inflammation. Current therapeutic strategies to treat airway disease include the use of muscarinic and leukotriene receptor antagonists; however, these pharmaceuticals demonstrate a limited clinical efficacy as multiple Gq-coupled receptor subtypes contribute to these pathologies. Thus, broadly inhibiting the activation of Gq may be an advantageous therapeutic approach. Here, we investigated the effects of broadly inhibiting Gq activation in vitro and ex vivo using receptor-dependent and receptor-independent strategies. P4pal-10 is a protease activated receptor 4-derived pepducin that exhibits efficacy toward multiple Gq-coupled receptors. Mechanistic studies demonstrated that P4pal-10 selectively inhibits all G protein coupling to several Gq-coupled receptors, including protease activated receptor 1, muscarinic acetylcholine M3, and histamine H1 receptors, while demonstrating no direct effect on Gq. We also evaluated the ability of FR900359, also known as UBO-QIC, to directly inhibit Gq activation. FR900359 inhibited spontaneous Gαq nucleotide exchange, while having little effect on Gαsβγ, Gαiβγ, or Gα12/13βγ heterotrimer activity. Both P4pal-10 and FR900359 inhibited Gq-mediated intracellular signaling and primary human airway smooth muscle growth, whereas only FR900359 effectively interdicted agonist-promoted airway contraction in human precision cut lung slices. These studies serve as a proof of concept that the broad-based inhibition of Gq activation may be a useful therapeutic approach to treat multiple common pathologies of airway disease. PMID:26464325

  17. Prothymosin alpha selectively enhances estrogen receptor transcriptional activity by interacting with a repressor of estrogen receptor activity.

    PubMed

    Martini, P G; Delage-Mourroux, R; Kraichely, D M; Katzenellenbogen, B S

    2000-09-01

    We find that prothymosin alpha (PTalpha) selectively enhances transcriptional activation by the estrogen receptor (ER) but not transcriptional activity of other nuclear hormone receptors. This selectivity for ER is explained by PTalpha interaction not with ER, but with a 37-kDa protein denoted REA, for repressor of estrogen receptor activity, a protein that we have previously shown binds to ER, blocking coactivator binding to ER. We isolated PTalpha, known to be a chromatin-remodeling protein associated with cell proliferation, using REA as bait in a yeast two-hybrid screen with a cDNA library from MCF-7 human breast cancer cells. PTalpha increases the magnitude of ERalpha transcriptional activity three- to fourfold. It shows lesser enhancement of ERbeta transcriptional activity and has no influence on the transcriptional activity of other nuclear hormone receptors (progesterone receptor, glucocorticoid receptor, thyroid hormone receptor, or retinoic acid receptor) or on the basal activity of ERs. In contrast, the steroid receptor coactivator SRC-1 increases transcriptional activity of all of these receptors. Cotransfection of PTalpha or SRC-1 with increasing amounts of REA, as well as competitive glutathione S-transferase pulldown and mammalian two-hybrid studies, show that REA competes with PTalpha (or SRC-1) for regulation of ER transcriptional activity and suppresses the ER stimulation by PTalpha or SRC-1, indicating that REA can function as an anticoactivator in cells. Our data support a model in which PTalpha, which does not interact with ER, selectively enhances the transcriptional activity of the ER but not that of other nuclear receptors by recruiting the repressive REA protein away from ER, thereby allowing effective coactivation of ER with SRC-1 or other coregulators. The ability of PTalpha to directly interact in vitro and in vivo with REA, a selective coregulator of the ER, thereby enabling the interaction of ER with coactivators, appears to explain

  18. Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

    PubMed

    Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2014-09-15

    Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 μg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists. PMID:25063794

  19. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    PubMed

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells. PMID:27500644

  20. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells

    PubMed Central

    Freund, Jacquelyn; May, Rebecca M.; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K.; Kambayashi, Taku

    2016-01-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells. PMID:27500644

  1. Chronic hyperammonemia induces tonic activation of NMDA receptors in cerebellum.

    PubMed

    ElMlili, Nisrin; Boix, Jordi; Ahabrach, Hanan; Rodrigo, Regina; Errami, Mohammed; Felipo, Vicente

    2010-02-01

    Reduced function of the glutamate--nitric oxide (NO)--cGMP pathway is responsible for some cognitive alterations in rats with hyperammonemia and hepatic encephalopathy. Hyperammonemia impairs the pathway in cerebellum by increasing neuronal nitric oxide synthase (nNOS) phosphorylation in Ser847 by calcium-calmodulin-dependent protein kinase II (CaMKII), reducing nNOS activity, and by reducing nNOS amount in synaptic membranes, which reduces its activation following NMDA receptors activation. The reason for increased CaMKII activity in hyperammonemia remains unknown. We hypothesized that it would be as a result of increased tonic activation of NMDA receptors. The aims of this work were to assess: (i) whether tonic NMDA activation receptors is increased in cerebellum in chronic hyperammonemia in vivo; and (ii) whether this tonic activation is responsible for increased CaMKII activity and reduced activity of nNOS and of the glutamate--NO--cGMP pathway. Blocking NMDA receptors with MK-801 increases cGMP and NO metabolites in cerebellum in vivo and in slices from hyperammonemic rats. This is because of reduced phosphorylation and activity of CaMKII, leading to normalization of nNOS phosphorylation and activity. MK-801 also increases nNOS in synaptic membranes and reduces it in cytosol. This indicates that hyperammonemia increases tonic activation of NMDA receptors leading to reduced activity of nNOS and of the glutamate--NO--cGMP pathway. PMID:20002515

  2. Biological activity of a polypeptide modulator of TRPV1 receptor.

    PubMed

    Dyachenko, I A; Andreev, Ya A; Logashina, Yu A; Murashev, A N; Grishin, E V

    2015-11-01

    This paper presents data on the activity of a new APHC2 polypeptide modulator of TRPV1 receptors, which was isolated from the sea anemone Heteractis crispa. It has been shown that APHC2 has an analgesic activity, does not impair normal motor activity, and does not change body temperature of experimental animals, which has a great practical value for design of potent analgesics of a new generation. Further study of the characteristics of binding of the polypeptide to the TRPV1 receptor may show approaches to the development of other antagonists of this receptor that do not influence the body temperature. PMID:26725234

  3. Human receptor activation by aroclor 1260, a polychlorinated biphenyl mixture.

    PubMed

    Wahlang, Banrida; Falkner, K Cameron; Clair, Heather B; Al-Eryani, Laila; Prough, Russell A; States, J Christopher; Coslo, Denise M; Omiecinski, Curtis J; Cave, Matthew C

    2014-08-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  4. Human Receptor Activation by Aroclor 1260, a Polychlorinated Biphenyl Mixture

    PubMed Central

    Wahlang, Banrida; Falkner, K. Cameron; Clair, Heather B.; Al-Eryani, Laila; Prough, Russell A.; States, J. Christopher; Coslo, Denise M.; Omiecinski, Curtis J.; Cave, Matthew C.

    2014-01-01

    Polychlorinated biphenyls (PCBs) are persistent environmental toxicants, present in 100% of U.S. adults and dose-dependently associated with obesity and non-alcoholic fatty liver disease (NAFLD). PCBs are predicted to interact with receptors previously implicated in xenobiotic/energy metabolism and NAFLD. These receptors include the aryl hydrocarbon receptor (AhR), pregnane xenobiotic receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptors (PPARs), liver-X-receptor (LXRα), and farnesoid-X-receptor (FXR). This study evaluates Aroclor 1260, a PCB mixture with congener composition mimicking that of human adipose tissue, and selected congeners, as potential ligands for these receptors utilizing human hepatoma-derived (HepG2) and primate-derived (COS-1) cell lines, and primary human hepatocytes. Aroclor 1260 (20 μg/ml) activated AhR, and PCB 126, a minor component, was a potent inducer. Aroclor 1260 activated PXR in a simple concentration-dependent manner at concentrations ≥10 μg/ml. Among the congeners tested, PCBs 138, 149, 151, 174, 183, 187, and 196 activated PXR. Aroclor 1260 activated CAR2 and CAR3 variants at lower concentrations and antagonize CAR2 activation by the CAR agonist, CITCO, at higher concentrations (≥20 μg/ml). Additionally, Aroclor 1260 induced CYP2B6 in primary hepatocytes. At subtoxic doses, Aroclor 1260 did not activate LXR or FXR and had no effect on LXR- or FXR-dependent induction by the agonists T0901317 or GW4064, respectively. Aroclor 1260 (20 μg/ml) suppressed PPARα activation by the agonist nafenopin, although none of the congeners tested demonstrated significant inhibition. The results suggest that Aroclor 1260 is a human AhR, PXR and CAR3 agonist, a mixed agonist/antagonist for CAR2, and an antagonist for human PPARα. PMID:24812009

  5. Characterization of peroxisome proliferator-activiated receptor alpha (PPARalpha)-independent effects of PPARalpha activators in the rodent liver: Di(2-ethylehexyl) phthalate activates the constitutive activated receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha). Recent studies indicate that the plasticizer di-2-ethylhexyl ph...

  6. Amyloid β peptide oligomers directly activate NMDA receptors.

    PubMed

    Texidó, Laura; Martín-Satué, Mireia; Alberdi, Elena; Solsona, Carles; Matute, Carlos

    2011-03-01

    Amyloid beta (Aβ) oligomers accumulate in the brain tissue of Alzheimer disease patients and are related to disease pathogenesis. The precise mechanisms by which Aβ oligomers cause neurotoxicity remain unknown. We recently reported that Aβ oligomers cause intracellular Ca(2+) overload and neuronal death that can be prevented by NMDA receptor antagonists. This study investigated whether Aβ oligomers directly activated NMDA receptors (NMDARs) using NR1/NR2A and NR1/NR2B receptors that were heterologously expressed in Xenopus laevis oocytes. Indeed, Aβ oligomers induced inward non-desensitizing currents that were blocked in the presence of the NMDA receptor antagonists memantine, APV, and MK-801. Intriguingly, the amplitude of the responses to Aβ oligomers was greater for NR1/NR2A heteromers than for NR1/NR2B heteromers expressed in oocytes. Consistent with these findings, we observed that the increase in the cytosolic concentration of Ca(2+) induced by Aβ oligomers in cortical neurons is prevented by AP5, a broad spectrum NMDA receptor antagonist, but slightly attenuated by ifenprodil which blocks receptors with the NR2B subunit. Together, these results indicate that Aβ oligomers directly activate NMDA receptors, particularly those with the NR2A subunit, and further suggest that drugs that attenuate the activity of such receptors may prevent Aβ damage to neurons in Alzheimeŕs disease. PMID:21349580

  7. Specific activation of the thyrotropin receptor by trypsin.

    PubMed

    Van Sande, J; Massart, C; Costagliola, S; Allgeier, A; Cetani, F; Vassart, G; Dumont, J E

    1996-05-31

    The identification of 16 different activating mutations in the TSH receptor, found in patients suffering from toxic autonomous adenomas or congenital hyperthyroidism, leads to the concept that this receptor is in a constrained conformation in its wild-type form. We used mild trypsin treatment of CHO-K1 cells or COS-7 cells, stably or transiently transfected with the human TSH receptor, respectively, and measured its consequences on the TSH receptor coupled cascades, i.e. cyclic AMP and inositol-phosphates accumulation. A 2-min, 0.01% trypsin treatment increased stably cyclic AMP but not inositol-phosphates formation. This was not observed after chymotrypsin, thrombin and endoproteinase glu C treatment. The TSH action on cyclic AMP was decreased by only 25%. The effect was also observed in cells expressing the dog TSH receptor. It was not observed in MSH receptor, LH receptor expressing or mock transfected cells (vector alone). It is therefore specific for the TSH receptor, for its action on the Gs/adenylate cyclase cascade, and for the proteolytic cleavage caused by trypsin. Using monoclonal (A. Johnstone and P. Shepherd, personal communication) and polyclonal antibodies directed against the extracellular domain of the TSH receptor, it was shown that treatment by trypsin removes or destroys a VFFEEQ epitope (residues 354-359) from the receptor. The effect mimics the action of TSH as it activates Gs alpha and enhances the action of forskolin. It is not reversible in 1 h. The results support the concept that activation of the receptor (by hormone, autoantibodies, mutations or mild proteolysis) might involve the relief of a built-in negative constrain. They suggest that the C-terminal portion of the large extracellular domain plays a role in the maintenance of this constrain. PMID:8807635

  8. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  9. Receptor tyrosine kinases: mechanisms of activation and signaling

    PubMed Central

    Hubbard, Stevan R.; Miller, W. Todd

    2008-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication. These single-pass transmembrane receptors, which bind polypeptide ligands — mainly growth factors — play key roles in processes such as cellular growth, differentiation, metabolism and motility. Recent progress has been achieved towards an understanding of the precise (and varied) mechanisms by which RTKs are activated by ligand binding and by which signals are propagated from the activated receptors to downstream targets in the cell. PMID:17306972

  10. Quantitative structure-activity relationship models with receptor-dependent descriptors for predicting peroxisome proliferator-activated receptor activities of thiazolidinedione and oxazolidinedione derivatives.

    PubMed

    Lather, Viney; Kairys, Visvaldas; Fernandes, Miguel X

    2009-04-01

    A quantitative structure-activity relationship study has been carried out, in which the relationship between the peroxisome proliferator-activated receptor alpha and the peroxisome proliferator-activated receptor gamma agonistic activities of thiazolidinedione and oxazolidinedione derivatives and quantitative descriptors, V(site) calculated in a receptor-dependent manner is modeled. These descriptors quantify the volume occupied by the optimized ligands in regions that are either common or specific to the superimposed binding sites of the targets under consideration. The quantitative structure-activity relationship models were built by forward stepwise linear regression modeling for a training set of 27 compounds and validated for a test set of seven compounds, resulting in a squared correlation coefficient value of 0.90 for peroxisome proliferator-activated receptor alpha and of 0.89 for peroxisome proliferator-activated receptor gamma. The leave-one-out cross-validation and test set predictability squared correlation coefficient values for these models were 0.85 and 0.62 for peroxisome proliferator-activated receptor alpha and 0.89 and 0.50 for peroxisome proliferator-activated receptor gamma respectively. A dual peroxisome proliferator-activated receptor model has also been developed, and it indicates the structural features required for the design of ligands with dual peroxisome proliferator-activated receptor activity. These quantitative structure-activity relationship models show the importance of the descriptors here introduced in the prediction and interpretation of the compounds affinity and selectivity. PMID:19243388

  11. Mincle suppresses Toll-like receptor 4 activation.

    PubMed

    Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

    2016-07-01

    Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. PMID:26747838

  12. Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins.

    PubMed

    Hay, Debbie L; Walker, Christopher S; Gingell, Joseph J; Ladds, Graham; Reynolds, Christopher A; Poyner, David R

    2016-04-15

    Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine. PMID:27068971

  13. Functions of the extracellular histidine residues of receptor activity-modifying proteins vary within adrenomedullin receptors

    SciTech Connect

    Kuwasako, Kenji Kitamura, Kazuo; Nagata, Sayaka; Kato, Johji

    2008-12-05

    Receptor activity-modifying protein (RAMP)-2 and -3 chaperone calcitonin receptor-like receptor (CRLR) to the plasma membrane, where together they form heterodimeric adrenomedullin (AM) receptors. We investigated the contributions made by His residues situated in the RAMP extracellular domain to AM receptor trafficking and receptor signaling by co-expressing hCRLR and V5-tagged-hRAMP2 or -3 mutants in which a His residue was substituted with Ala in HEK-293 cells. Flow cytometric analysis revealed that hRAMP2-H71A mediated normal hCRLR surface delivery, but the resultant heterodimers showed significantly diminished [{sup 125}I]AM binding and AM-evoked cAMP production. Expression of hRAMP2-H124A and -H127A impaired surface delivery of hCRLR, which impaired or abolishing AM binding and receptor signaling. Although hRAMP3-H97A mediated full surface delivery of hCRLR, the resultant heterodimers showed impaired AM binding and signaling. Other His residues appeared uninvolved in hCRLR-related functions. Thus, the His residues of hRAMP2 and -3 differentially govern AM receptor function.

  14. Activation of G protein by opioid receptors: role of receptor number and G-protein concentration.

    PubMed

    Remmers, A E; Clark, M J; Alt, A; Medzihradsky, F; Woods, J H; Traynor, J R

    2000-05-19

    The collision-coupling model for receptor-G-protein interaction predicts that the rate of G-protein activation is dependent on receptor density, but not G-protein levels. C6 cells expressing mu- or delta-opioid receptors, or SH-SY5Y cells, were treated with beta-funaltrexamine (mu) or naltrindole-5'-isothiocyanate (delta) to decrease receptor number. The time course of full or partial agonist-stimulated ¿35SGTPgammaS binding did not vary in C6 cell membranes containing <1-25 pmol/mg mu-opioid receptor, or 1. 4-4.3 pmol/mg delta-opioid receptor, or in SHSY5Y cells containing 0. 16-0.39 pmol/mg receptor. The association of ¿35SGTPgammaS binding was faster in membranes from C6mu cells than from C6delta cells. A 10-fold reduction in functional G-protein, following pertussis toxin treatment, lowered the maximal level of ¿35SGTPgammaS binding but not the association rate. These data indicate a compartmentalization of opioid receptors and G protein at the cell membrane. PMID:10822058

  15. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  16. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

  17. A dual mechanism for impairment of GABAA receptor activity by NMDA receptor activation in rat cerebellum granule cells.

    PubMed

    Robello, M; Amico, C; Cupello, A

    1997-01-01

    The function of the GABAA receptor has been studied using the whole cell voltage clamp recording technique in rat cerebellum granule cells in culture. Activation of NMDA-type glutamate receptors causes a reduction in the effect of GABA. Full GABAA receptor activity was recovered after washing out NMDA and NMDA action was prevented in a Mg+2 containing medium. The NMDA effect was also absent when extracellular Ca+2 was replaced by Ba+2 and when 10 mM Bapta was present in the intracellular solution. Charge accumulations via voltage activated Ca+2 channels greater than the ones via NMDA receptors do not cause any reduction in GABAA receptor function, suggesting that Ca+2 influx through NMDA receptor channels is critical for the effect. The NMDA effect was reduced by including adenosine-5'-O-3-thiophosphate (ATP-gamma-S) in the internal solution and there was a reduction in the NMDA effect caused by deltamethrin, a calcineurin inhibitor. Part of the NMDA induced GABAA receptor impairment was prevented by prior treatment with L-arginine. Analogously, part of the NMDA effect was prevented by blockage of NO-synthase activity by N omega-nitro-L-arginine. A combination of NO-synthase and calcineurin inhibitors completely eliminated the NMDA action. An analogous result was obtained by combining the NO-synthase inhibitor with the addition of ATP-gamma-S to the pipette medium. The additivity of the prevention of the NMDA impairment of GABAA receptor by blocking the L-arginine/NO pathway and inhibiting calcineurin activity suggests an independent involvement of these two pathways in the interaction between NMDA and the GABAA receptor. On the one hand Ca+2 influx across NMDA channels activates calcineurin and dephosphorylates the GABAA receptor complex directly or dephosphorylates proteins critical for the function of the receptor. On the other hand, Ca+2 influx activates NO-synthase and induces nitric oxide production, which regulates such receptors via protein kinase G

  18. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    PubMed Central

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors and protease-activated receptors, both of which are GPCRs, are central to the metastatic melanoma phenotype and may serve as potential targets in novel therapies against melanoma and other cancers. PMID:27379162

  19. Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

    PubMed Central

    Pandhare, Akash; Grozdanov, Petar N.; Jansen, Michaela

    2016-01-01

    In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap. PMID:27045630

  20. Clinically used selective oestrogen receptor modulators increase LDL receptor activity in primary human lymphocytes

    PubMed Central

    Cerrato, F; Fernández-Suárez, M E; Alonso, R; Alonso, M; Vázquez, C; Pastor, O; Mata, P; Lasunción, M A; Gómez-Coronado, D

    2015-01-01

    Background and Purpose Treatment with selective oestrogen receptor modulators (SERMs) reduces low-density lipoprotein (LDL) cholesterol levels. We assessed the effect of tamoxifen, raloxifene and toremifene and their combinations with lovastatin on LDL receptor activity in lymphocytes from normolipidaemic and familial hypercholesterolaemic (FH) subjects, and human HepG2 hepatocytes and MOLT-4 lymphoblasts. Experimental Approach Lymphocytes were isolated from peripheral blood, treated with different compounds, and 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanine perchlorate (DiI)-labelled LDL uptake was analysed by flow cytometry. Key Results Tamoxifen, toremifene and raloxifene, in this order, stimulated DiI-LDL uptake by lymphocytes by inhibiting LDL-derived cholesterol trafficking and subsequent down-regulation of LDL receptor expression. Differently to what occurred in HepG2 and MOLT-4 cells, only tamoxifen consistently displayed a potentiating effect with lovastatin in primary lymphocytes. The SERM-mediated increase in LDL receptor activity was not altered by the anti-oestrogen ICI 182 780 nor was it reproduced by 17β-oestradiol. However, the tamoxifen-active metabolite endoxifen was equally effective as tamoxifen. The SERMs produced similar effects on LDL receptor activity in heterozygous FH lymphocytes as in normal lymphocytes, although none of them had a potentiating effect with lovastatin in heterozygous FH lymphocytes. The SERMs had no effect in homozygous FH lymphocytes. Conclusions and Implications Clinically used SERMs up-regulate LDL receptors in primary human lymphocytes. There is a mild enhancement between SERMs and lovastatin of lymphocyte LDLR activity, the potentiation being greater in HepG2 and MOLT-4 cells. The effect of SERMs is independent of oestrogen receptors but is preserved in the tamoxifen-active metabolite endoxifen. This mechanism may contribute to the cholesterol-lowering action of SERMs. PMID:25395200

  1. Transcriptional activation of nuclear estrogen receptor and progesterone receptor and its regulation.

    PubMed

    Xin, Qi-Liang; Qiu, Jing-Tao; Cui, Sheng; Xia, Guo-Liang; Wang, Hai-Bin

    2016-08-25

    Estrogen receptor (ER) and progesterone receptor (PR) are two important members of steroid receptors family, an evolutionarily conserved family of transcription factors. Upon binding to their ligands, ER and PR enter cell nucleus to interact with specific DNA element in the context of chromatin to initiate the transcription of diverse target genes, which largely depends on the timely recruitment of a wide range of cofactors. Moreover, the interactions between steroid hormones and their respective receptors also trigger post-translational modifications on these receptors to fine-tune their transcriptional activities. Besides the well-known phosphorylation modifications on tyrosine and serine/threonine residues, recent studies have identified several other covalent modifications, such as ubiquitylation and sumoylation. These post-translational modifications of steroid receptors affect its stability, subcellular localization, and/or cofactor recruitment; eventually influence the duration and extent of transcriptional activation. This review is to focus on the recent research progress on the transcriptional activation of nuclear ER and PR as well as their physiological functions in early pregnancy, which may help us to better understand related female reproductive diseases. PMID:27546504

  2. Analyzing the activation of the melanocortin-2 receptor of tetrapods.

    PubMed

    Dores, Robert M; Liang, Liang

    2014-07-01

    Following the biochemical characterization of the pituitary hormone, adrenocorticotropin (ACTH), in the 1950's, a number of structure/function studies were done which identifies two amino acid motifs in ACTH, the HFRW motif and KKRR motif, as critical for the activation of the "ACTH" receptor on adrenal cortex cells. In the 1990's the "ACTH" receptor was identified as a member of the melanocortin receptor gene family, and given the name melanocortin-2 receptor (MC2R). Since that time a number of studies on both tetrapod and teleost MC2R orthologs have established that these orthologs can only be activated by ACTH, but not by any of the MSH-sized melanocortin ligands, and these orthologs require interaction with the melanocortin-2 receptor accessory protein (MRAP) for functional expression. This review summarizes recent structure/function studies on human ACTH, and points out the importance of the GKPVG motif in ACTH for the activation of the receptor. In this regard, a multiple-step model for the activation of tetrapod and teleost MC2R orthologs is presented, and the evolution of gnathostome MC2R ligand selectivity and the requirement for MRAP interaction is discussed in light of a recent study on a cartilaginous fish MC2R ortholog. This review contains excerpts from the Gorbman/Bern Lecture presented at the Second Meeting of the North American Society for Comparative Endocrinology (NASCE). PMID:24713445

  3. Monitoring leptin activity using the chicken leptin receptor.

    PubMed

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold. PMID:18434362

  4. Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid-Activated Receptor

    SciTech Connect

    Kruse, Schoen W; Suino-Powell, Kelly; Zhou, X Edward; Kretschman, Jennifer E; Reynolds, Ross; Vonrhein, Clemens; Xu, Yong; Wang, Liliang; Tsai, Sophia Y; Tsai, Ming-Jer; Xu, H Eric

    2010-01-12

    The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 {angstrom} crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix {alpha}10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

  5. The biologically active conformations of ligand CCK B receptor

    NASA Astrophysics Data System (ADS)

    Kuznetsov, Pavel E.; Kuznetsova, Nina B.; Schulgin, Sergey V.; Rogacheva, Svetlana M.; Sinyakov, Valeriy V.; Kovtun, Viktor A.

    2006-07-01

    We analyzed literature data about structures of ligands of CCK B receptor. The structure of the binding site (fragments of the third extracellular loop and the seventh transmembrane helix of CCK B receptor) was determined recently by experiments. We were finding presumable biologically active conformations (BAC) of the ligands by two methods. One of them is based on the fact that the most stable conformations of the biologically active peptide on the phase interface "water-lipophilic medium" are often similar to the BAC. Another method is based on the formation of the stable ligand-receptor complex during the modeling procedure. We used Monte-Carlo method with the fixed geometry of the receptor and the optimized geometry of tetrapeptide cholecystokinin (CCK-4). It has been shown, that the first method should be used to find BAC of antagonists of CCK B receptor. The strategy of the formation of the stable ligand-receptor complex during the modeling procedure can be used for the designing of peptide agonists of CCK B receptor.

  6. Opportunistic activation of TRP receptors by endogenous lipids: Exploiting lipidomics to understand TRP receptor cellular communication

    PubMed Central

    Bradshaw, Heather B.; Raboune, Siham; Hollis, Jennifer L.

    2012-01-01

    Transient receptor potential channels (TRPs) form a large family of ubiquitous non-selective cation channels that function as cellular sensors and in many cases regulate intracellular calcium. Identification of the endogenous ligands that activate these TRP receptors is still under intense investigation with the majority of these channels still remaining “orphans”. That these channels respond to a variety of external stimuli (e.g. plant-derived lipids, changes in temperature, and changes in pH) provides a framework for their abilities as cellular sensors, however, the mechanism of direct activation is still under much debate and research. In the cases where endogenous ligands (predominately lipids) have shown direct activation of a channel, multiple ligands have been shown to activate the same channel suggesting that these receptors are “promiscuous” in nature. Lipidomics of a growing class of endogenous lipids, N-acyl amides, the most famous of which is N-arachidonoyl ethanolamine (the endogenous cannabinoid, Anandamide) is providing a novel set of ligands that have been shown to activate some members of the TRP family and have the potential to deorphanize many more. Here it is argued that activation of TRPV receptors, a subset of the larger family of TRPs, by multiple endogenous lipids that are structurally analogous is a model system to drive our understanding that many TRP receptors are not promiscuous, but are more characteristically “opportunistic” in nature; exploiting the structural similarity and biosynthesis of a narrow range of analogous endogenous lipids. In addition, this manuscript will compare the activation properties of TRPC5 to the activity profile of an “orphan” lipid, N-palmitoyl glycine; further demonstrating that lipidomics aimed at expanding our knowledge of the family of N-acyl amides has the potential to provide novel avenues of research for TRP receptors. PMID:23178153

  7. Flavonoids as dietary regulators of nuclear receptor activity

    PubMed Central

    Avior, Yishai; Bomze, David; Ramon, Ory

    2013-01-01

    Metabolic diseases such as obesity, type II diabetes, and dyslipidemia are a rising cause of mortality worldwide. The progression of many metabolic diseases is fundamentally regulated on the transcriptional level by a family of ligand-activated transcription factors, called nuclear receptors, which detect and respond to metabolic changes. Their role in maintaining metabolic homeostasis makes nuclear receptors an important pharmaceutical and dietary target. This review will present the growing evidence that flavonoids, natural secondary plant metabolites, are important regulators of nuclear receptor activity. Structural similarities between flavonoids and cholesterol derivatives combined with the promiscuous nature of most nuclear receptors provide a wealth of possibilities for pharmaceutical and dietary modulation of metabolism. While the challenges of bringing flavonoid-derived therapeutics to the market are significant, we consider this rapidly growing field to be an essential aspect of the functional food initiative and an important mine for pharmaceutical compounds. PMID:23598551

  8. Ligands for the Nuclear Peroxisome Proliferator-Activated Receptor Gamma.

    PubMed

    Sauer, Sascha

    2015-10-01

    Nuclear receptors are ligand-activated transcription factors, which represent a primary class of drug targets. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a key player in various biological processes. PPARγ is widely known as the target protein of the thiazolidinediones for treating type 2 diabetes. Moreover, PPARγ ligands can induce anti-inflammatory and potentially additional beneficial effects. Recent mechanistic insights of PPARγ modulation give hope the next generation of efficient PPARγ-based drugs with fewer side effects can be developed. Furthermore, chemical approaches that make use of synergistic action of combinatorial ligands are promising alternatives for providing tailored medicine. Lessons learned from fine-tuning the action of PPARγ can provide avenues for efficient molecular intervention via many other nuclear receptors to combat common diseases. PMID:26435213

  9. Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors

    PubMed Central

    Terunuma, Miho; Vargas, Karina J.; Wilkins, Megan E.; Ramírez, Omar A.; Jaureguiberry-Bravo, Matías; Pangalos, Menelas N.; Smart, Trevor G.; Moss, Stephen J.; Couve, Andrés

    2010-01-01

    Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors. PMID:20643948

  10. Toll-like receptors 2 and 4 exert opposite effects on the contractile response induced by serotonin in mouse colon: role of serotonin receptors.

    PubMed

    Forcén, R; Latorre, E; Pardo, J; Alcalde, A I; Murillo, M D; Grasa, L

    2016-08-01

    What is the central question of this study? The action of Toll-like receptors (TLRs) 2 and 4 on the motor response to serotonin in mouse colon has not previously been reported. What is the main finding and its importance? Toll-like receptors 2 and 4 modulate the serotonin-induced contractile response in mouse colon by modifying the expression of serotonin (5-HT) receptors. Alterations in 5-HT2A and 5-HT2C receptors explain the increase of the response to serotonin in TLR2(-/-) mice. Alterations in 5-HT2C and 5-HT4 receptors explain the suppression of the response to serotonin in TLR4(-/-) mice. The microbiota, through Toll-like receptors (TLRs), may regulate gastrointestinal motility by activating neuroendocrine mechanisms. We evaluated the influence of TLR2 and TLR4 in spontaneous contractions and in the serotonin (5-HT)-induced motor response in mouse colon, and assessed the 5-HT receptors involved. Muscle contractility studies to evaluate the intestinal spontaneous motility and the response to 5-HT were performed in the colon from wild-type (WT), TLR2(-/-) , TLR4(-/-) and TLR2/4 double knockout (DKO) mice. The 5-HT receptor mRNA expression was determined by real-time PCR. The amplitude and frequency of the spontaneous contractions of the colon were smaller in TLR4(-/-) and TLR2/4 DKO mice with respect to WT mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 100 μm 5-HT evoked a contractile response. The contractile response induced by 5-HT was significantly higher in TLR2(-/-) than in WT mice. In TLR4(-/-) mice, 5-HT did not evoke any contractile response. The mRNA expression of 5-HT2A was increased in TLR2(-/-) and TLR2/4 DKO mice. The 5-HT2C and 5-HT4 mRNA expressions were increased in TLR4(-/-) and TLR2/4 DKO mice. The 5-HT2C mRNA expression was diminished in TLR2(-/-) mice. The 5-HT3 mRNA expression was increased in TLR2(-/-) , TLR4(-/-) and TLR2/4 DKO mice. The 5-HT7 mRNA expression was diminished in TLR2/4 DKO mice. In WT, TLR2(-/-) and TLR2/4 DKO mice, 5-HT2

  11. Allosteric Activation of a G Protein-coupled Receptor with Cell-penetrating Receptor Mimetics*

    PubMed Central

    Zhang, Ping; Leger, Andrew J.; Baleja, James D.; Rana, Rajashree; Corlin, Tiffany; Nguyen, Nga; Koukos, Georgios; Bohm, Andrew; Covic, Lidija; Kuliopulos, Athan

    2015-01-01

    G protein-coupled receptors (GPCRs) are remarkably versatile signaling systems that are activated by a large number of different agonists on the outside of the cell. However, the inside surface of the receptors that couple to G proteins has not yet been effectively modulated for activity or treatment of diseases. Pepducins are cell-penetrating lipopeptides that have enabled chemical and physical access to the intracellular face of GPCRs. The structure of a third intracellular (i3) loop agonist, pepducin, based on protease-activated receptor-1 (PAR1) was solved by NMR and found to closely resemble the i3 loop structure predicted for the intact receptor in the on-state. Mechanistic studies revealed that the pepducin directly interacts with the intracellular H8 helix region of PAR1 and allosterically activates the receptor through the adjacent (D/N)PXXYYY motif through a dimer-like mechanism. The i3 pepducin enhances PAR1/Gα subunit interactions and induces a conformational change in fluorescently labeled PAR1 in a very similar manner to that induced by thrombin. As pepducins can potentially be made to target any GPCR, these data provide insight into the identification of allosteric modulators to this major drug target class. PMID:25934391

  12. N-Benzylpiperidine Derivatives as α7 Nicotinic Receptor Antagonists.

    PubMed

    Criado, Manuel; Mulet, José; Sala, Francisco; Sala, Salvador; Colmena, Inés; Gandía, Luis; Bautista-Aguilera, Oscar M; Samadi, Abdelouahid; Chioua, Mourad; Marco-Contelles, José

    2016-08-17

    A series of multitarget directed propargylamines, as well as other differently susbstituted piperidines have been screened as potential modulators of neuronal nicotinic acetylcholine receptors (nAChRs). Most of them showed antagonist actions on α7 nAChRs. Especially, compounds 13, 26, and 38 displayed submicromolar IC50 values on homomeric α7 nAChRs, whereas they were less effective on heteromeric α3β4 and α4β2 nAChRs (up to 20-fold higher IC50 values in the case of 13). Antagonism was concentration dependent and noncompetitive, suggesting that these compounds behave as negative allosteric modulators of nAChRs. Upon the study of a series of less complex derivatives, the N-benzylpiperidine motif, common to these compounds, was found to be the main pharmacophoric group. Thus, 2-(1-benzylpiperidin-4-yl)-ethylamine (48) showed an inhibitory potency comparable to the one of the previous compounds and also a clear preference for α7 nAChRs. In a neuroblastoma cell line, representative compounds 13 and 48 also inhibited, in a concentration-dependent manner, cytosolic Ca(2+) signals mediated by nAChRs. Finally, compounds 38 and 13 inhibited 5-HT3A serotonin receptors whereas they had no effect on α1 glycine receptors. Given the multifactorial nature of many pathologies in which nAChRs are involved, these piperidine antagonists could have a therapeutic potential in cases where cholinergic activity has to be negatively modulated. PMID:27254782

  13. Structural mechanism of glutamate receptor activation and desensitization.

    PubMed

    Meyerson, Joel R; Kumar, Janesh; Chittori, Sagar; Rao, Prashant; Pierson, Jason; Bartesaghi, Alberto; Mayer, Mark L; Subramaniam, Sriram

    2014-10-16

    Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a 'corkscrew' motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating. PMID:25119039

  14. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    PubMed Central

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with GPCRs redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch. PMID:26216096

  15. In vitro screening of major neurotransmitter systems possibly involved in the mechanism of action of antibodies to S100 protein in released-active form

    PubMed Central

    Gorbunov, Evgeniy A; Ertuzun, Irina A; Kachaeva, Evgeniya V; Tarasov, Sergey A; Epstein, Oleg I

    2015-01-01

    Experimentally and clinically, it was shown that released-active form of antibodies to S100 protein (RAF of Abs to S100) exerts a wide range of pharmacological activities: anxiolytic, antiasthenic, antiaggressive, stress-protective, antihypoxic, antiischemic, neuroprotective, and nootropic. The purpose of this study was to determine the influence of RAF of Abs to S100 on major neurotransmitter systems (serotoninergic, GABAergic, dopaminergic, and on sigma receptors as well) which are possibly involved in its mechanism of pharmacological activity. Radioligand binding assays were used for assessment of the drug influence on ligand–receptor interaction. [35S]GTPγS binding assay, cyclic adenosine monophosphate HTRF™, cellular dielectric spectroscopy assays, and assays based on measurement of intracellular concentration of Ca2+ ions were used for assessment of agonist or antagonist properties of the drug toward receptors. RAF of Abs to S100 increased radioligand binding to 5-HT1F, 5-HT2B, 5-HT2Cedited, 5-HT3, and to D3 receptors by 142.0%, 131.9%, 149.3%, 120.7%, and 126.3%, respectively. Also, the drug significantly inhibited specific binding of radioligands to GABAB1A/B2 receptors by 25.8%, and to both native and recombinant human sigma1 receptors by 75.3% and 40.32%, respectively. In the functional assays, it was shown that the drug exerted antagonism at 5-HT1B, D3, and GABAB1A/B2 receptors inhibiting agonist-induced responses by 23.24%, 32.76%, and 30.2%, respectively. On the contrary, the drug exerted an agonist effect at 5-HT1A receptors enhancing receptor functional activity by 28.0%. The pharmacological profiling of RAF of Abs to S100 among 27 receptor provides evidence for drug-related modification of major neurotransmitter systems. PMID:26604768

  16. Interfering with mineralocorticoid receptor activation: the past, present, and future

    PubMed Central

    2014-01-01

    Aldosterone is a potent mineralocorticoid produced by the adrenal gland. Aldosterone binds to and activates the mineralocorticoid receptor (MR) in a plethora of tissues, but the cardiovascular actions of aldosterone are of primary interest clinically. Although MR antagonists were developed as antihypertensive agents, they are now considered to be important therapeutic options for patients with heart failure. Specifically, blocking only the MR has proven to be a difficult task because of its similarity to other steroid receptors, including the androgen and progesterone receptors. This lack of specificity caused the use of the first-generation mineralocorticoid receptor antagonists to be fraught with difficulty because of the side effects produced by drug administration. However, in recent years, several advances have been made that could potentially increase the clinical use of agents that inhibit the actions of aldosterone. These will be discussed here along with some examples of the beneficial effects of these new therapeutic agents. PMID:25165560

  17. Regulation of Proteome Maintenance Gene Expression by Activators of Peroxisome Proliferator-Activated Receptor a (PPARa)

    EPA Science Inventory

    The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) is activated by a large number of xenobiotic and hypolipidemic compounds called peroxisome proliferator chemicals (PPC). One agonist of PPARa (WY-14,643) regulates responses in the mouse liver to chemic...

  18. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  19. Biologic activity of antigen receptors artificially incorporated onto B lymphocytes.

    PubMed

    Peacock, J S; Londo, T R; Roess, D A; Barisas, B G

    1986-09-15

    We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1

  20. Liver X Receptor and Peroxisome Proliferator-Activated Receptor Agonist from Cornus alternifolia

    PubMed Central

    He, Yang-Qing; Ma, Guo-Yi; Peng, Jiang-nan; Ma, Zhan-Ying; Hamann, Mark T.

    2012-01-01

    Background Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptors superfamily and are transcription factors activated by specific ligands. Liver X receptors (LXR) belong to the nuclear hormone receptors and have been shown to play an important role in cholesterol homeostasis. From the previous screening of several medicinal plants for potential partial PPARγ agonists, the extracts of Cornus alternifolia were found to exhibit promising bioactivity. In this paper, we report the isolation and structural elucidation of four new compounds and their potential as ligands for PPAR. Methods The new compounds were extracted from the leaves of Cornus alternifolia and fractionated by high-performance liquid chromatography. Their structures were elucidated on the basis of spectroscopic evidence and analysis of their hydrolysis products. Results Three new iridoid glycosides including an iridolactone, alternosides A-C (1–3), a new megastigmane glycoside, cornalternoside (4) and 10 known compounds, were obtained from the leaves of Cornus alternifolia. Kaempferol-3-O-β-glucopyranoside (5) exhibited potent agonistic activities for PPARα, PPARγ and LXR with EC50 values of 0.62, 3.0 and 1.8 μ M, respectively. Conclusions We isolated four new and ten known compounds from Cornus alternifolia, and one known compound showed agonistic activities for PPARα, PPARγ and LXR. General significance Compound 1 is the first example of a naturally occurring iridoid glycoside containing a β-glucopyranoside moiety at C-6. PMID:22353334

  1. A Phytochrome Sensory Domain Permits Receptor Activation by Red Light.

    PubMed

    Reichhart, Eva; Ingles-Prieto, Alvaro; Tichy, Alexandra-Madelaine; McKenzie, Catherine; Janovjak, Harald

    2016-05-17

    Optogenetics and photopharmacology enable the spatio-temporal control of cell and animal behavior by light. Although red light offers deep-tissue penetration and minimal phototoxicity, very few red-light-sensitive optogenetic methods are currently available. We have now developed a red-light-induced homodimerization domain. We first showed that an optimized sensory domain of the cyanobacterial phytochrome 1 can be expressed robustly and without cytotoxicity in human cells. We then applied this domain to induce the dimerization of two receptor tyrosine kinases-the fibroblast growth factor receptor 1 and the neurotrophin receptor trkB. This new optogenetic method was then used to activate the MAPK/ERK pathway non-invasively in mammalian tissue and in multicolor cell-signaling experiments. The light-controlled dimerizer and red-light-activated receptor tyrosine kinases will prove useful to regulate a variety of cellular processes with light. PMID:27101018

  2. Structural basis for selective activation of ABA receptors

    SciTech Connect

    Peterson, Francis C.; Burgie, E. Sethe; Park, Sang-Youl; Jensen, Davin R.; Weiner, Joshua J.; Bingman, Craig A.; Chang, Chia-En A.; Cutler, Sean R.; Phillips, Jr., George N.; Volkman, Brian F.

    2010-11-01

    Changing environmental conditions and lessening fresh water supplies have sparked intense interest in understanding and manipulating abscisic acid (ABA) signaling, which controls adaptive responses to drought and other abiotic stressors. We recently discovered a selective ABA agonist, pyrabactin, and used it to discover its primary target PYR1, the founding member of the PYR/PYL family of soluble ABA receptors. To understand pyrabactin's selectivity, we have taken a combined structural, chemical and genetic approach. We show that subtle differences between receptor binding pockets control ligand orientation between productive and nonproductive modes. Nonproductive binding occurs without gate closure and prevents receptor activation. Observations in solution show that these orientations are in rapid equilibrium that can be shifted by mutations to control maximal agonist activity. Our results provide a robust framework for the design of new agonists and reveal a new mechanism for agonist selectivity.

  3. Novel positive allosteric modulators of GABAA receptors with anesthetic activity

    PubMed Central

    Maldifassi, Maria C.; Baur, Roland; Pierce, David; Nourmahnad, Anahita; Forman, Stuart A.; Sigel, Erwin

    2016-01-01

    GABAA receptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2 GABAA receptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2 GABAA receptors expressed in Xenopus laevis oocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α− subunit transmembrane interfaces of the GABAA receptors. We then used concatenated receptors to dissect the involvement of individual β+/α− interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model, Xenopus laevis tadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics. PMID:27198062

  4. Positive allosteric modulators of α7 nicotinic acetylcholine receptors affect neither the function of other ligand- and voltage-gated ion channels and acetylcholinesterase, nor β-amyloid content.

    PubMed

    Arias, Hugo R; Ravazzini, Federica; Targowska-Duda, Katarzyna M; Kaczor, Agnieszka A; Feuerbach, Dominik; Boffi, Juan C; Draczkowski, Piotr; Montag, Dirk; Brown, Brandon M; Elgoyhen, Ana Belén; Jozwiak, Krzysztof; Puia, Giulia

    2016-07-01

    The activity of positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (AChRs), including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and 3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) α7, rat (r) α9α10, hα3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] and voltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and β-amyloid (Aβ) content. The functional results indicate that PAM-2 inhibits hα3-containing AChRs (IC50=26±6μM) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitive GluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE, and PAM-4 does not affect agonist-activated rα9α10 AChRs. Relevant clinical concentrations of PAM-2-4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 and GluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of Aβ42 in an Alzheimer's disease mouse model (i.e., 5XFAD). The molecular docking and dynamics results using the hα7 model suggest that the active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. These results support our previous study showing that these PAMs are selective for the α7 AChR, and clarify that the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets. PMID:27129924

  5. Endocannabinoid tone versus constitutive activity of cannabinoid receptors

    PubMed Central

    Howlett, Allyn C; Reggio, Patricia H; Childers, Steven R; Hampson, Robert E; Ulloa, Nadine M; Deutsch, Dale G

    2011-01-01

    This review evaluates the cellular mechanisms of constitutive activity of the cannabinoid (CB) receptors, its reversal by inverse agonists, and discusses the pitfalls and problems in the interpretation of the research data. The notion is presented that endogenously produced anandamide (AEA) and 2-arachidonoylglycerol (2-AG) serve as autocrine or paracrine stimulators of the CB receptors, giving the appearance of constitutive activity. It is proposed that one cannot interpret inverse agonist studies without inference to the receptors' environment vis-à-vis the endocannabinoid agonists which themselves are highly lipophilic compounds with a preference for membranes. The endocannabinoid tone is governed by a combination of synthetic pathways and inactivation involving transport and degradation. The synthesis and degradation of 2-AG is well characterized, and 2-AG has been strongly implicated in retrograde signalling in neurons. Data implicating endocannabinoids in paracrine regulation have been described. Endocannabinoid ligands can traverse the cell's interior and potentially be stored on fatty acid-binding proteins (FABPs). Molecular modelling predicts that the endocannabinoids derived from membrane phospholipids can laterally diffuse to enter the CB receptor from the lipid bilayer. Considering that endocannabinoid signalling to CB receptors is a much more likely scenario than is receptor activation in the absence of agonist ligands, researchers are advised to refrain from assuming constitutive activity except for experimental models known to be devoid of endocannabinoid ligands. LINKED ARTICLES This article is part of a themed issue on Cannabinoids in Biology and Medicine. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2011.163.issue-7 PMID:21545414

  6. Peroxisome proliferator-activated receptors in the cardiovascular system

    PubMed Central

    Bishop-Bailey, David

    2000-01-01

    Peroxisome proliferator-activated receptor (PPAR)s are a family of three nuclear hormone receptors, PPARα, -δ, and -γ, which are members of the steriod receptor superfamily. The first member of the family (PPARα) was originally discovered as the mediator by which a number of xenobiotic drugs cause peroxisome proliferation in the liver. Defined functions for all these receptors, until recently, mainly concerned their ability to regulate energy balance, with PPARα being involved in β-oxidation pathways, and PPARγ in the differentiation of adipocytes. Little is known about the functions of PPARδ, though it is the most ubiquitously expressed. Since their discovery, PPARs have been shown to be expressed in monocytes/macrophages, the heart, vascular smooth muscle cells, endothelial cells, and in atherosclerotic lesions. Furthermore, PPARs can be activated by a vast number of compounds including synthetic drugs, of the clofibrate, and anti-diabetic thiazoldinedione classes, polyunsaturated fatty acids, and a number of eicosanoids, including prostaglandins, lipoxygenase products, and oxidized low density lipoprotein. This review will aim to introduce the field of PPAR nuclear hormone receptors, and discuss the discovery and actions of PPARs in the cardiovascular system, as well as the source of potential ligands. PMID:10696077

  7. Interaction of the C-terminal acidic domain of the insulin receptor with histone modulates the receptor kinase activity.

    PubMed

    Baron, V; Kaliman, P; Alengrin, F; Van Obberghen, E

    1995-04-01

    In this study, we investigated the role of the insulin receptor domain 1270-1280, an acid-rich sequence located in the receptor C-terminus. Antipeptide IgG raised against this sequence were obtained and used to analyze their effect on receptor function. Antipeptide IgG inhibited receptor autophosphorylation at Tyr1146, Tyr1150 and Tyr1151. These sites are known to be key modulators of the receptor activity. Autophosphorylation at other sites may also have been inhibited. The antipeptide antibody decreased the receptor kinase activity measured with poly(Glu80Tyr20) and a synthetic peptide corresponding to the proreceptor sequence 1142-1158. We provide evidence that the effect of the antibody on substrate phosphorylation may result from the control of the phosphorylation level of the receptor. Concerning the action of the antipeptide IgG on the receptor kinase activity, histone did not behave similarly to poly(Glu80Tyr20). The antibody recognizing sequence 1270-1280 competed with histone for an overlapping binding site. Histone also modulated insulin receptor autophosphorylation, supporting the idea that interference with domain 1270-1280 alters the receptor kinase. Our data suggest that the acidic region including residues 1270-1280 of the insulin receptor C-terminus is involved in the following events: (a) receptor binding with histone, an exogenous substrate of the receptor kinase, and (b) the regulation of receptor autophosphorylation and kinase activity. Based on these observations, we would like to propose that this insulin receptor domain could interact with cellular proteins modulating the receptor kinase. PMID:7744039

  8. OX1 orexin/hypocretin receptor activation of phospholipase D

    PubMed Central

    Jäntti, MH; Putula, J; Somerharju, P; Frohman, MA; Kukkonen, JP

    2012-01-01

    BACKGROUND AND PURPOSE Orexin receptors potently signal to lipid messenger systems, and our previous studies have suggested that PLD would be one of these. We thus wanted to verify this by direct measurements and clarify the molecular mechanism of the coupling. EXPERIMENTAL APPROACH Orexin receptor-mediated PLD activation was investigated in CHO cells stably expressing human OX1 orexin receptors using [14C]-oleic acid-prelabelling and the transphosphatidylation assay. KEY RESULTS Orexin stimulation strongly increased PLD activity – even more so than the phorbol ester TPA (12-O-tetradecanoyl-phorbol-13-acetate), a highly potent activator of PLD. Both orexin and TPA responses were mediated by PLD1. Orexin-A and -B showed approximately 10-fold difference in potency, and the concentration–response curves were biphasic. Using pharmacological inhibitors and activators, both orexin and TPA were shown to signal to PLD1 via the novel PKC isoform, PKCδ. In contrast, pharmacological or molecular biological inhibitors of Rho family proteins RhoA/B/C, cdc42 and Rac did not inhibit the orexin (or the TPA) response, nor did the molecular biological inhibitors of PKD. In addition, neither cAMP elevation, Gαi/o nor Gβγ seemed to play an important role in the orexin response. CONCLUSIONS AND IMPLICATIONS Stimulation of OX1 receptors potently activates PLD (probably PLD1) in CHO cells and this is mediated by PKCδ but not other PKC isoforms, PKDs or Rho family G-proteins. At present, the physiological significance of orexin-induced PLD activation is unknown, but this is not the first time we have identified PKCδ in orexin signalling, and thus some specific signalling cascade may exist between orexin receptors and PKCδ. PMID:21718304

  9. Covalent agonists for studying G protein-coupled receptor activation

    PubMed Central

    Weichert, Dietmar; Kruse, Andrew C.; Manglik, Aashish; Hiller, Christine; Zhang, Cheng; Hübner, Harald; Kobilka, Brian K.; Gmeiner, Peter

    2014-01-01

    Structural studies on G protein-coupled receptors (GPCRs) provide important insights into the architecture and function of these important drug targets. However, the crystallization of GPCRs in active states is particularly challenging, requiring the formation of stable and conformationally homogeneous ligand-receptor complexes. Native hormones, neurotransmitters, and synthetic agonists that bind with low affinity are ineffective at stabilizing an active state for crystallogenesis. To promote structural studies on the pharmacologically highly relevant class of aminergic GPCRs, we here present the development of covalently binding molecular tools activating Gs-, Gi-, and Gq-coupled receptors. The covalent agonists are derived from the monoamine neurotransmitters noradrenaline, dopamine, serotonin, and histamine, and they were accessed using a general and versatile synthetic strategy. We demonstrate that the tool compounds presented herein display an efficient covalent binding mode and that the respective covalent ligand-receptor complexes activate G proteins comparable to the natural neurotransmitters. A crystal structure of the β2-adrenoreceptor in complex with a covalent noradrenaline analog and a conformationally selective antibody (nanobody) verified that these agonists can be used to facilitate crystallogenesis. PMID:25006259

  10. The cardiovascular effects of peroxisome proliferator-activated receptor agonists.

    PubMed

    Friedland, Sayuri N; Leong, Aaron; Filion, Kristian B; Genest, Jacques; Lega, Iliana C; Mottillo, Salvatore; Poirier, Paul; Reoch, Jennifer; Eisenberg, Mark J

    2012-02-01

    Although peroxisome proliferator-activated receptor agonists are prescribed to improve cardiovascular risk factors, their cardiovascular safety is controversial. We therefore reviewed the literature to identify landmark randomized controlled trials evaluating the effect of peroxisome proliferator-activated receptor gamma agonists (pioglitazone and rosiglitazone), alpha agonists (fenofibrate and gemfibrozil), and pan agonists (bezafibrate, muraglitazar, ragaglitazar, tesaglitazar, and aleglitazar) on cardiovascular outcomes. Pioglitazone may modestly reduce cardiovascular events but also may increase the risk of bladder cancer. Rosiglitazone increases the risk of myocardial infarction and has been withdrawn in European and restricted in the United States. Fibrates improve cardiovascular outcomes only in select subgroups: fenofibrate in diabetic patients with metabolic syndrome, gemfibrozil in patients with dyslipidemia, and bezafibrate in patients with diabetes or metabolic syndrome. The cardiovascular safety of the new pan agonist aleglitazar, currently in phase II trials, remains to be determined. The heterogenous effects of peroxisome proliferator-activated receptor agonists to date highlight the importance of postmarketing surveillance. The critical question of why peroxisome proliferator-activated receptor agonists seem to improve cardiovascular risk factors without significantly improving cardiovascular outcomes requires further investigation. PMID:22269613

  11. Nuclear Receptor Activity and Liver Cancer Lesion Progression

    EPA Science Inventory

    Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPARα,...

  12. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  13. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

    PubMed

    Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

    2014-07-01

    Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

  14. Lysophosphatidylserine analogues differentially activate three LysoPS receptors.

    PubMed

    Uwamizu, Akiharu; Inoue, Asuka; Suzuki, Kensuke; Okudaira, Michiyo; Shuto, Akira; Shinjo, Yuji; Ishiguro, Jun; Makide, Kumiko; Ikubo, Masaya; Nakamura, Sho; Jung, Sejin; Sayama, Misa; Otani, Yuko; Ohwada, Tomohiko; Aoki, Junken

    2015-03-01

    Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors. PMID:25320102

  15. Receptor antagonism/agonism can be uncoupled from pharmacoperone activity.

    PubMed

    Janovick, Jo Ann; Spicer, Timothy P; Smith, Emery; Bannister, Thomas D; Kenakin, Terry; Scampavia, Louis; Conn, P Michael

    2016-10-15

    Pharmacoperones rescue misrouted mutants of the vasopressin receptor type 2 (V2R) and enable them to traffic to the correct biological locus where they function. Previously, a library of nearly 645,000 structures was interrogated with a high throughput screen; pharmacoperones were identified for V2R mutants with a view toward correcting the underlying mutational defects in nephrogenic diabetes insipidus. In the present study, an orthologous assay was used to evaluate hits from the earlier study. We found no consistent relation between antagonism or agonism and pharmacoperone activity. Active pharmacoperones were identified which had minimal antagonistic activity. This increases the therapeutic reach of these drugs, since virtually all pharmacoperone drugs reported to date were selected from peptidomimetic antagonists. Such mixed-activity drugs have a complex pharmacology limiting their therapeutic utility and requiring their removal prior to stimulation of the receptor with agonist. PMID:27389877

  16. Mechanisms of NOD-like receptor-associated inflammasome activation.

    PubMed

    Wen, Haitao; Miao, Edward A; Ting, Jenny P-Y

    2013-09-19

    A major function of a subfamily of NLR (nucleotide-binding domain, leucine-rich repeat containing, or NOD-like receptor) proteins is in inflammasome activation, which has been implicated in a multitude of disease models and human diseases. This work will highlight key progress in understanding the mechanisms that activate the best-studied NLRs (NLRP3, NLRC4, NAIP, and NLRP1) and in uncovering inflammasome NLRs. PMID:24054327

  17. Adenosine kinase inhibitors attenuate opiate withdrawal via adenosine receptor activation.

    PubMed

    Kaplan, G B; Coyle, T S

    1998-11-27

    Previous studies have demonstrated a role for adenosine in mediating opiate effects. This study examines the effects of indirect activation of adenosine receptors, via treatment with adenosine kinase inhibitors, on the expression of opiate withdrawal in mice. Mice receive chronic morphine treatment via implantation of subcutaneous morphine pellets (75 mg) for 72 h. Mice then receive parenteral treatment with adenosine kinase inhibitors, either 5'-amino-5'-deoxyadenosine (2, 5, 20, 40 mg/kg, intraperitoneal or i.p.) or iodotubericidin (1, 2, 5 mg/kg, i.p.), followed by naloxone injection and opiate withdrawal signs are measured over 20 min. Both adenosine kinase inhibitors significantly reduce the following opiate withdrawal signs in a dose-dependent manner compared to vehicle: withdrawal jumps, teeth chattering, forepaw tremors, and forepaw treads. Additionally, 5'-amino-5'-deoxyadenosine significantly reduces withdrawal-induced diarrhea and weight loss. Effects of 5'-amino-5'-deoxyadenosine (40 mg/kg) on opiate withdrawal signs appear to be mediated via adenosine receptor activation as they are reversed by pretreatment by adenosine receptor antagonist caffeine (20 mg, i.p.) but not by selective phosphodiesterase inhibitor Ro 20-1724 (10 mg/kg, i.p.). Adenosine receptor activation via adenosine kinase inhibitor treatment attenuates opiate withdrawal and these agents may be generally useful in the treatment of drug withdrawal syndromes. PMID:9865523

  18. Receptor Activity-Modifying Proteins (RAMPs): New Insights and Roles.

    PubMed

    Hay, Debbie L; Pioszak, Augen A

    2016-01-01

    It is now recognized that G protein-coupled receptors (GPCRs), once considered largely independent functional units, have a far more diverse molecular architecture. Receptor activity-modifying proteins (RAMPs) provide an important example of proteins that interact with GPCRs to modify their function. RAMPs are able to act as pharmacological switches and chaperones, and they can regulate signaling and/or trafficking in a receptor-dependent manner. This review covers recent discoveries in the RAMP field and summarizes the known GPCR partners and functions of RAMPs. We also discuss the first peptide-bound structures of RAMP-GPCR complexes, which give insight into the molecular mechanisms that enable RAMPs to alter the pharmacology and signaling of GPCRs. PMID:26514202

  19. Interaction of receptor-activity-modifying protein1 with tubulin.

    PubMed

    Kunz, Thomas H; Mueller-Steiner, Sarah; Schwerdtfeger, Kerstin; Kleinert, Peter; Troxler, Heinz; Kelm, Jens M; Ittner, Lars M; Fischer, Jan A; Born, Walter

    2007-08-01

    Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors. PMID:17493758

  20. A review of granisetron, 5-hydroxytryptamine3 receptor antagonists, and other antiemetics.

    PubMed

    Hsu, Eric S

    2010-01-01

    Nausea and vomiting are 2 of the most upsetting adverse reactions of chemotherapy. Current guidelines propose 5-hydroxytryptamine3 (5-HT3) receptor antagonists as a pharmacologic intervention for acute and delayed nausea and vomiting [chemotherapy-induced nausea and vomiting (CINV)] associated with moderately and highly emetogenic chemotherapy. Meanwhile, both postoperative nausea and vomiting (PONV) and postdischarge nausea and vomiting are challenging situations after surgeries and procedures. Prophylactic and therapeutic combinations of antiemetics are recommended in patients at high risk of suffering from PONV and postdischarge nausea and vomiting. Granisetron (Kytril) is a selective 5-HT3 receptor antagonist that does not induce or inhibit the hepatic cytochrome P-450 system in vitro. There are also 4 other antagonists of 5-HT3 receptor (dolasetron, ondansetron, palonosetron, and tropisetron) being metabolized via the CYP2D6 and are subject to potential genetic polymorphism. The launch of a new class of antiemetics, the substance P/neurokinin1 receptor antagonists, was attributed to the scientific update on the central generator responsible for emesis and role of substance P. There has been mounting interest in exploring integrative medicine, either acupuncture or acustimulation of P6 (Nei-Kuwan), to complement the western medicine for prevention and management of nausea and vomiting. The potential application of cannabinoids, either alone or in combination with other agents of different mechanism, could contribute further to improve outcome in CINV. Implementation of future treatment guidelines for more effective management of CINV and PONV could certainly improve the efficacy and outcome of cancer and postoperative care. PMID:20844345

  1. CINPA1 Is an Inhibitor of Constitutive Androstane Receptor That Does Not Activate Pregnane X Receptor

    PubMed Central

    Cherian, Milu T; Lin, Wenwei; Wu, Jing

    2015-01-01

    Constitutive androstane receptor (CAR) and pregnane X receptor (PXR) are xenobiotic sensors that enhance the detoxification and elimination of xenobiotics and endobiotics by modulating the expression of genes encoding drug-metabolizing enzymes and transporters. Elevated levels of drug-metabolizing enzymes and efflux transporters, resulting from CAR activation in various cancers, promote the elimination of chemotherapeutic agents, leading to reduced therapeutic effectiveness and acquired drug resistance. CAR inhibitors, in combination with existing chemotherapeutics, could therefore be used to attenuate multidrug resistance in cancers. Interestingly, all previously reported CAR inverse-agonists are also activators of PXR, rendering them mechanistically counterproductive in tissues where both these xenobiotic receptors are present and active. We used a directed high-throughput screening approach, followed by subsequent mechanistic studies, to identify novel, potent, and specific small-molecule CAR inhibitors that do not activate PXR. We describe here one such inhibitor, CINPA1 (CAR inhibitor not PXR activator 1), capable of reducing CAR-mediated transcription with an IC50 of ∼70 nM. CINPA1 1) is a specific xenobiotic receptor inhibitor and has no cytotoxic effects up to 30 µM; 2) inhibits CAR-mediated gene expression in primary human hepatocytes, where CAR is endogenously expressed; 3) does not alter the protein levels or subcellular localization of CAR; 4) increases corepressor and reduces coactivator interaction with the CAR ligand-binding domain in mammalian two-hybrid assays; and 5) disrupts CAR binding to the promoter regions of target genes in chromatin immunoprecipitation assays. CINPA1 could be used as a novel molecular tool for understanding CAR function. PMID:25762023

  2. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    PubMed

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand. PMID:25916672

  3. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  4. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity.

    PubMed

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-12-15

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser(696) and Ser(698) in the JM (juxtamembrane) region and probably Ser(886) and/or Ser(893) in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser(717) in the JM, and at Ser(733), Thr(752), Ser(783), Ser(864), Ser(911), Ser(958) and Thr(998) in the kinase domain. The LC-ESI-MS/MS spectra provided support that up to three sites (Thr(890), Ser(893) and Thr(894)) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr(890), Ser(893), Thr(894) and Thr(899), differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  5. Allosteric receptor activation by the plant peptide hormone phytosulfokine.

    PubMed

    Wang, Jizong; Li, Hongju; Han, Zhifu; Zhang, Heqiao; Wang, Tong; Lin, Guangzhong; Chang, Junbiao; Yang, Weicai; Chai, Jijie

    2015-09-10

    Phytosulfokine (PSK) is a disulfated pentapeptide that has a ubiquitous role in plant growth and development. PSK is perceived by its receptor PSKR, a leucine-rich repeat receptor kinase (LRR-RK). The mechanisms underlying the recognition of PSK, the activation of PSKR and the identity of the components downstream of the initial binding remain elusive. Here we report the crystal structures of the extracellular LRR domain of PSKR in free, PSK- and co-receptor-bound forms. The structures reveal that PSK interacts mainly with a β-strand from the island domain of PSKR, forming an anti-β-sheet. The two sulfate moieties of PSK interact directly with PSKR, sensitizing PSKR recognition of PSK. Supported by biochemical, structural and genetic evidence, PSK binding enhances PSKR heterodimerization with the somatic embryogenesis receptor-like kinases (SERKs). However, PSK is not directly involved in PSKR-SERK interaction but stabilizes PSKR island domain for recruitment of a SERK. Our data reveal the structural basis for PSKR recognition of PSK and allosteric activation of PSKR by PSK, opening up new avenues for the design of PSKR-specific small molecules. PMID:26308901

  6. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages

    PubMed Central

    Moreira-Souza, Aline Cristina Abreu; Marinho, Ygor; Correa, Gladys; Santoro, Giani França; Coutinho, Claudia Mara Lara Melo; Vommaro, Rossiane Claudia; Coutinho-Silva, Robson

    2015-01-01

    Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane – subdivided into P2Y and P2X subfamilies - whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection. PMID:26192447

  7. The Molecular Mechanism of P2Y1 Receptor Activation.

    PubMed

    Yuan, Shuguang; Chan, H C Stephen; Vogel, Horst; Filipek, Slawomir; Stevens, Raymond C; Palczewski, Krzysztof

    2016-08-22

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1 R) is activated by adenosine 5'-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1 R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 μs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1 R activation. PMID:27460867

  8. The Molecular Mechanism of P2Y1 Receptor Activation

    PubMed Central

    Chan, H. C. Stephen; Vogel, Horst; Filipek, Slawomir

    2016-01-01

    Human purinergic G protein-coupled receptor P2Y1 (P2Y1R) is activated by adenosine 5’-diphosphate (ADP) to induce platelet activation and thereby serves as an important antithrombotic drug target. Crystal structures of P2Y1R revealed that one ligand (MRS2500) binds to the extracellular vestibule of this GPCR, whereas another (BPTU) occupies the surface between transmembrane (TM) helices TM2 and TM3. We introduced a total of 20 µs all-atom long-timescale molecular dynamic (MD) simulations to inquire why two molecules in completely different locations both serve as antagonists while ADP activates the receptor. Our results indicate that BPTU acts as an antagonist by stabilizing extracellular helix bundles leading to an increase of the lipid order, whereas MRS2500 blocks signaling by occupying the ligand binding site. Both antagonists stabilize an ionic lock within the receptor. However, binding of ADP breaks this ionic lock, forming a continuous water channel that leads to P2Y1R activation. PMID:27460867

  9. Neurosteroid Structure-Activity Relationships for Functional Activation of Extrasynaptic δGABA(A) Receptors.

    PubMed

    Carver, Chase Matthew; Reddy, Doodipala Samba

    2016-04-01

    Synaptic GABAA receptors are primary mediators of rapid inhibition in the brain and play a key role in the pathophysiology of epilepsy and other neurologic disorders. The δ-subunit GABAA receptors are expressed extrasynaptically in the dentate gyrus and contribute to tonic inhibition, promoting network shunting as well as reducing seizure susceptibility. However, the neurosteroid structure-function relationship at δGABA(A) receptors within the native hippocampus neurons remains unclear. Here we report a structure-activity relationship for neurosteroid modulation of extrasynaptic GABAA receptor-mediated tonic inhibition in the murine dentate gyrus granule cells. We recorded neurosteroid allosteric potentiation of GABA as well as direct activation of tonic currents using a wide array of natural and synthetic neurosteroids. Our results shows that, for all neurosteroids, the C3α-OH group remains obligatory for extrasynaptic receptor functional activity, as C3β-OH epimers were inactive in activating tonic currents. Allopregnanolone and related pregnane analogs exhibited the highest potency and maximal efficacy in promoting tonic currents. Alterations at the C17 or C20 region of the neurosteroid molecule drastically altered the transduction kinetics of tonic current activation. The androstane analogs had the weakest modulatory response among the analogs tested. Neurosteroid potentiation of tonic currents was completely (approximately 95%) diminished in granule cells from δ-knockout mice, suggesting that δ-subunit receptors are essential for neurosteroid activity. The neurosteroid sensitivity of δGABA(A) receptors was confirmed at the systems level using a 6-Hz seizure test. A consensus neurosteroid pharmacophore model at extrasynaptic δGABA(A) receptors is proposed based on a structure-activity relationship for activation of tonic current and seizure protection. PMID:26857959

  10. An allosteric role for receptor activity-modifying proteins in defining GPCR pharmacology

    PubMed Central

    J Gingell, Joseph; Simms, John; Barwell, James; Poyner, David R; Watkins, Harriet A; Pioszak, Augen A; Sexton, Patrick M; Hay, Debbie L

    2016-01-01

    G protein-coupled receptors are allosteric proteins that control transmission of external signals to regulate cellular response. Although agonist binding promotes canonical G protein signalling transmitted through conformational changes, G protein-coupled receptors also interact with other proteins. These include other G protein-coupled receptors, other receptors and channels, regulatory proteins and receptor-modifying proteins, notably receptor activity-modifying proteins (RAMPs). RAMPs have at least 11 G protein-coupled receptor partners, including many class B G protein-coupled receptors. Prototypic is the calcitonin receptor, with altered ligand specificity when co-expressed with RAMPs. To gain molecular insight into the consequences of this protein–protein interaction, we combined molecular modelling with mutagenesis of the calcitonin receptor extracellular domain, assessed in ligand binding and functional assays. Although some calcitonin receptor residues are universally important for peptide interactions (calcitonin, amylin and calcitonin gene-related peptide) in calcitonin receptor alone or with receptor activity-modifying protein, others have RAMP-dependent effects, whereby mutations decreased amylin/calcitonin gene-related peptide potency substantially only when RAMP was present. Remarkably, the key residues were completely conserved between calcitonin receptor and AMY receptors, and between subtypes of AMY receptor that have different ligand preferences. Mutations at the interface between calcitonin receptor and RAMP affected ligand pharmacology in a RAMP-dependent manner, suggesting that RAMP may allosterically influence the calcitonin receptor conformation. Supporting this, molecular dynamics simulations suggested that the calcitonin receptor extracellular N-terminal domain is more flexible in the presence of receptor activity-modifying protein 1. Thus, RAMPs may act in an allosteric manner to generate a spectrum of unique calcitonin receptor

  11. Human peroxisome proliferator-activated receptor mRNA and protein expression during development

    EPA Science Inventory

    The peroxisome proliferator-activated receptors (PPAR) are nuclear hormone receptors that regulate lipid and glucose homeostasis and are important in reproduction and development. PPARs are targets ofpharmaceuticals and are also activated by environmental contaminants, including ...

  12. Mammalian EGF receptor activation by the rhomboid protease RHBDL2.

    PubMed

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-05-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  13. Mammalian EGF receptor activation by the rhomboid protease RHBDL2

    PubMed Central

    Adrain, Colin; Strisovsky, Kvido; Zettl, Markus; Hu, Landian; Lemberg, Marius K; Freeman, Matthew

    2011-01-01

    The epidermal growth factor receptor (EGFR) has several functions in mammalian development and disease, particularly cancer. Most EGF ligands are synthesized as membrane-tethered precursors, and their proteolytic release activates signalling. In Drosophila, rhomboid intramembrane proteases catalyse the release of EGF-family ligands; however, in mammals this seems to be primarily achieved by ADAM-family metalloproteases. We report here that EGF is an efficient substrate of the mammalian rhomboid RHBDL2. RHBDL2 cleaves EGF just outside its transmembrane domain, thereby facilitating its secretion and triggering activation of the EGFR. We have identified endogenous RHBDL2 activity in several tumour cell lines. PMID:21494248

  14. Bisphenol A and Its Analogues Activate Human Pregnane X Receptor

    PubMed Central

    Sui, Yipeng; Ai, Ni; Park, Se-Hyung; Rios-Pilier, Jennifer; Perkins, Jordan T.; Welsh, William J.

    2012-01-01

    Background: Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA and its analogues are present in environmental and human samples. Many endocrine-disrupting chemicals, including BPA, have been shown to activate the pregnane X receptor (PXR), a nuclear receptor that functions as a master regulator of xenobiotic metabolism. However, the detailed mechanism by which these chemicals activate PXR remains unknown. Objective: We investigated the mechanism by which BPA interacts with and activates PXR and examined selected BPA analogues to determine whether they bind to and activate PXR. Methods: Cell-based reporter assays, in silico ligand–PXR docking studies, and site-directed mutagenesis were combined to study the interaction between BPA and PXR. We also investigated the influence of BPA and its analogues on the regulation of PXR target genes in human LS180 cells. Results: We found that BPA and several of its analogues are potent agonists for human PXR (hPXR) but do not affect mouse PXR activity. We identified key residues within hPXR’s ligand-binding pocket that constitute points of interaction with BPA. We also deduced the structural requirements of BPA analogues that activate hPXR. BPA and its analogues can also induce PXR target gene expression in human LS180 cells. Conclusions: The present study advances our understanding of the mechanism by which BPA interacts with and activates human PXR. Activation of PXR by BPA may explain some of the adverse effects of BPA in humans. PMID:22214767

  15. Selective antagonism of the GABAA receptor by ciprofloxacin and biphenylacetic acid

    PubMed Central

    Green, M A; Halliwell, R F

    1997-01-01

    Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit γ-aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique. GABA (50 μM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3–10 μM) and picrotoxin (0.3–10 μM), with IC50 values and 95% confidence intervals (CI) of 1.2 μM (1.1–1.4) and 3.6 μM (3.0–4.3), respectively, and were potentiated by sodium pentobarbitone (30 μM) and diazepam (1 μM) to (mean±s.e.mean) 168±18% and 117±4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 μM)-evoked responses were inhibited by MDL 72222 (1 μM) to 10±4% of control; DMPP (10 μM)-evoked responses were inhibited by hexamethonium (100 μM) to 12±5% of control, and αbMeATP (30 μM)-evoked responses were inhibited by PPADS (10 μM) to 21±5% of control. Together, these data are consistent with activation of GABAA, 5-HT3, nicotinic ACh and P2X receptors, respectively. Ciprofloxacin (10–3000 μM) inhibited GABAA-mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 μM (148–275). BPAA (1–1000 μM) had little or no effect on the GABAA-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times. Responses mediated by 5-HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 μM), BPAA (100 μM) or the combination of these drugs (both at 100 μM). GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 μM (2.8–4.5), a value not significantly different from that determined in the vagus

  16. Selective antagonism of the GABA(A) receptor by ciprofloxacin and biphenylacetic acid.

    PubMed

    Green, M A; Halliwell, R F

    1997-10-01

    1. Previous studies have shown that ciprofloxacin and biphenylacetic acid (BPAA) synergistically inhibit y-aminobutyric acid (GABA)A receptors. In the present study, we have investigated the actions of these two drugs on other neuronal ligand-gated ion channels. 2. Agonist-evoked depolarizations were recorded from rat vagus and optic nerves in vitro by use of an extracellular recording technique. 3. GABA (50 microM)-evoked responses, in the vagus nerve in vitro, were inhibited by bicuculline (0.3-10 microM) and picrotoxin (0.3-10 microM), with IC50 values and 95% confidence intervals (CI) of 1.2 microM (1.1-1.4) and 3.6 microM (3.0-4.3), respectively, and were potentiated by sodium pentobarbitone (30 microM) and diazepam (1 microM) to (mean+/-s.e.mean) 168+/-18% and 117+/-4% of control, respectively. 5-Hydroxytryptamine (5-HT; 0.5 microM)-evoked responses were inhibited by MDL 72222 (1 microM) to 10+/-4% of control; DMPP (10 microM)-evoked responses were inhibited by hexamethonium (100 microM) to 12+/-5% of control, and alphabetaMeATP (30 microM)-evoked responses were inhibited by PPADS (10 microM) to 21+/-5% of control. Together, these data are consistent with activation of GABA(A), 5-HT3, nicotinic ACh and P2X receptors, respectively. 4 Ciprofloxacin (10-3000 microM) inhibited GABA(A)-mediated responses in the vagus nerve with an IC50 (and 95% CI) of 202 microM (148-275). BPAA (1-1000 microM) had little or no effect on the GABA(A)-mediated response but concentration-dependently potentiated the effects of ciprofloxacin by up to 33,000 times. 5. Responses mediated by 5-HT3, nicotinic ACh and P2X receptors in the vagus nerve and strychnine-sensitive glycine receptors in the optic nerve were little or unaffected by ciprofloxacin (100 microM), BPAA (100 microM) or the combination of these drugs (both at 100 microM). 6. GABA (1 mM)-evoked responses in the optic nerve were inhibited by bicuculline with an IC50 of 3.6 microM (2.8-4.5), a value not significantly different

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

    SciTech Connect

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

    2000-05-01

    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.

  18. Allosterism and Structure in Thermally Activated Transient Receptor Potential Channels.

    PubMed

    Diaz-Franulic, Ignacio; Poblete, Horacio; Miño-Galaz, Germán; González, Carlos; Latorre, Ramón

    2016-07-01

    The molecular sensors that mediate temperature changes in living organisms are a large family of proteins known as thermosensitive transient receptor potential (TRP) ion channels. These membrane proteins are polymodal receptors that can be activated by cold or hot temperatures, depending on the channel subtype, voltage, and ligands. The stimuli sensors are allosterically coupled to a pore domain, increasing the probability of finding the channel in its ion conductive conformation. In this review we first discuss the allosteric coupling between the temperature and voltage sensor modules and the pore domain, and then discuss the thermodynamic foundations of thermo-TRP channel activation. We provide a structural overview of the molecular determinants of temperature sensing. We also posit an anisotropic thermal diffusion model that may explain the large temperature sensitivity of TRP channels. Additionally, we examine the effect of several ligands on TRP channel function and the evidence regarding their mechanisms of action. PMID:27297398

  19. Manipulation of P2X Receptor Activities by Light Stimulation

    PubMed Central

    Kim, Sang Seong

    2016-01-01

    P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels. PMID:26884649

  20. Activation of the chicken gonadotropin-inhibitory hormone receptor reduces gonadotropin releasing hormone receptor signaling.

    PubMed

    Shimizu, Mamiko; Bédécarrats, Grégoy Y

    2010-06-01

    Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic peptide from the RFamide peptide family that has been identified in multiple avian species. Although GnIH has clearly been shown to reduce LH release from the anterior pituitary gland, its mechanism of action remains to be determined. The overall objectives of this study were (1) to characterize the GnIH receptor (GnIH-R) signaling pathway, (2) to evaluate potential interactions with gonadotropin releasing hormone type III receptor (GnRH-R-III) signaling, and (3) to determine the molecular mechanisms by which GnIH and GnRH regulate pituitary gonadotrope function during a reproductive cycle in the chicken. Using real-time PCR, we showed that in the chicken pituitary gland, GnIH-R mRNA levels fluctuate in an opposite manner to GnRH-R-III, with higher and lower levels observed during inactive and active reproductive stages, respectively. We demonstrated that the chicken GnIH-R signals by inhibiting adenylyl cyclase cAMP production, most likely by coupling to G(alphai). We also showed that this inhibition is sufficient to significantly reduce GnRH-induced cAMP responsive element (CRE) activation in a dose-dependent manner, and that the ratio of GnRH/GnIH receptors is a significant factor. We propose that in avian species, sexual maturation is characterized by a change in GnIH/GnRH receptor ratio, resulting in a switch in pituitary sensitivity from inhibitory (involving GnIH) to stimulatory (involving GnRH). In turn, decreasing GnIH-R signaling, combined with increasing GnRH-R-III signaling, results in significant increases in CRE activation, possibly initiating gonadotropin synthesis. PMID:20350548

  1. Activation of signalling by the activin receptor complex.

    PubMed Central

    Attisano, L; Wrana, J L; Montalvo, E; Massagué, J

    1996-01-01

    Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals. PMID:8622651

  2. Structural insights into μ-opioid receptor activation

    PubMed Central

    Huang, Weijiao; Manglik, Aashish; Venkatakrishnan, A. J.; Laeremans, Toon; Feinberg, Evan N.; Sanborn, Adrian L.; Kato, Hideaki E.; Livingston, Kathryn E.; Thorsen, Thor S.; Kling, Ralf; Granier, Sébastien; Gmeiner, Peter; Husbands, Stephen M.; Traynor, John R.; Weis, William I.; Steyaert, Jan; Dror, Ron O.; Kobilka, Brian K.

    2015-01-01

    Summary Activation of the μ-opioid receptor (μOR) is responsible for the efficacy of the most effective analgesics. To understand the structural basis for μOR activation, we obtained a 2.1 Å X-ray crystal structure of the μOR bound to the morphinan agonist BU72 and stabilized by a G protein-mimetic camelid-antibody fragment. The BU72-stabilized changes in the μOR binding pocket are subtle and differ from those observed for agonist-bound structures of the β2 adrenergic receptor (β2AR) and the M2 muscarinic receptor (M2R). Comparison with active β2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the μOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three GPCRs. PMID:26245379

  3. Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis

    PubMed Central

    Lin, Kuan-Hung; Ho, Ya-Hsuan; Chiang, Jui-Chung; Li, Meng-Wei; Lin, Shi-Hung; Chen, Wei-Min; Chiang, Chi-Ling; Lin, Yu-Nung; Yang, Ya-Jan; Chen, Chiung-Nien; Lu, Jenher; Huang, Chang-Jen; Tigyi, Gabor; Yao, Chao-Ling; Lee, Hsinyu

    2016-01-01

    Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34+ human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis. PMID:27244685

  4. Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis.

    PubMed

    Lin, Kuan-Hung; Ho, Ya-Hsuan; Chiang, Jui-Chung; Li, Meng-Wei; Lin, Shi-Hung; Chen, Wei-Min; Chiang, Chi-Ling; Lin, Yu-Nung; Yang, Ya-Jan; Chen, Chiung-Nien; Lu, Jenher; Huang, Chang-Jen; Tigyi, Gabor; Yao, Chao-Ling; Lee, Hsinyu

    2016-01-01

    Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34(+) human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis. PMID:27244685

  5. Ultrastructural and biochemical analysis of fibrinogen receptors on activated thrombocytes

    SciTech Connect

    O'Toole, E.T.

    1989-01-01

    The present studies have been concerned with the role of fibrinogen and its receptor, GP IIb/IIIa, during the activation and early aggregation of pigeon thrombocytes. Thrombocytes were surface labeled with {sup 125}I then separated on SDS-PAGE. Analysis by gel autoradiography revealed major bands at MW 145 kd and 98 kd, which corresponded to human GPIIb and GPIIIa. Immunologic similarity of the pigeon and human receptor components was established by dot blot analysis using polyclonal antibodies directed against human GPIIb and GPIIIa. Pigeon fibrinogen, isolated by plasma precipitation with PEG-1000 and purified over Sepharose 4B, was used to study receptor-ligand interaction. Separation of pigeon fibrinogen on SDS-PAGE resulted in three peptides having apparent MW of 62kd, 55kd, and 47kd which are comparable to human fibrinogen. Further similarity of human and pigeon fibrinogen was verified by immonodiffusion against an antibody specific for the human protein. The role of fibrinogen and its receptor in thrombocyte function was established by turbidimetric aggregation using thrombin as an agonist under conditions requiring Ca++ and fibrinogen.

  6. Dynamic correlation networks in human peroxisome proliferator-activated receptor-γ nuclear receptor protein.

    PubMed

    Fidelak, Jeremy; Ferrer, Silvia; Oberlin, Michael; Moras, Dino; Dejaegere, Annick; Stote, Roland H

    2010-10-01

    Peroxisome proliferator-activated receptor-γ nuclear receptor (PPAR-γ) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-γ. Here we use normal-mode analysis of PPAR-γ to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals. PMID:20496064

  7. Propofol Restores Transient Receptor Potential Vanilloid Receptor Subtype-1 Sensitivity via Activation of Transient Receptor Potential Ankyrin Receptor Subtype-1 in Sensory Neurons

    PubMed Central

    Zhang, Hongyu; Wickley, Peter J.; Sinha, Sayantani; Bratz, Ian N.; Damron, Derek S.

    2011-01-01

    Background Crosstalk between peripheral nociceptors belonging to the transient receptor potential vanilloid receptor subtype-1 (TRPV1) and ankyrin subtype-1 (TRPA1) family has recently been demonstrated. Moreover, the intravenous anesthetic propofol has been shown to directly activate TRPA1 receptors, and indirectly restore sensitivity of TRPV1 receptors in dorsal root ganglion (DRG) sensory neurons. Our objective was to determine the extent to which TRPA1 activation is involved in mediating the propofol-induced restoration of TRPV1 sensitivity. Methods Mouse DRG neurons were isolated by enzymatic dissociation and grown for 24 h. F-11 cells were transfected with complementary DNA for both TRPV1 and TRPA1 or TRPV1 only. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. Following TRPV1 de-sensitization with capsaicin (100 nM), cells were treated with propofol (1, 5 and 10 μM) alone, propofol in the presence of the TRPA1 antagonist, HC-030031 (0.5 μM) or the TRPA1 agonist, Allyl isothiocyanate (AITC, 100 μM) and capsaicin was then reapplied. Results In DRG neurons that contain both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in DRG neurons containing only TRPV1 receptors, exposure to propofol or AITC following de-sensitization did not restore capsaicin-induced TRPV1 sensitivity. Similarly, in F-11 cells transfected with both TRPV1 and TRPA1, propofol and AITC restored TRPV1 sensitivity. However, in F-11 cells transfected with TRPV1 only, neither propofol nor AITC were capable of restoring TRPV1 sensitivity. Conclusions These data demonstrate that propofol restores TRPV1 sensitivity in primary DRG neurons and in cultured F-11 cells transfected with both the TRPV1 and TRPA1 receptors via a TRPA1-dependent process. Propofol’s effects on sensory neurons may be clinically important and contribute to peripheral sensitization to nociceptive stimuli in traumatized tissue. PMID:21364461

  8. Differential effect of meclizine on the activity of human pregnane X receptor and constitutive androstane receptor.

    PubMed

    Lau, Aik Jiang; Yang, Guixiang; Rajaraman, Ganesh; Baucom, Christie C; Chang, Thomas K H

    2011-03-01

    Conflicting data exist as to whether meclizine is an activator of human pregnane X receptor (hPXR). Therefore, we conducted a detailed, systematic investigation to determine whether meclizine affects hPXR activity by performing a cell-based reporter gene assay, a time-resolved fluorescence resonance energy transfer competitive ligand-binding assay, a mammalian two-hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. In pregnane X receptor (PXR)-transfected HepG2 cells, meclizine activated hPXR to a greater extent than rat PXR. It bound to hPXR ligand-binding domain and recruited steroid receptor coactivator-1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene expression (CYP3A4) in human hepatocytes. However, it did not increase but decreased testosterone 6β-hydroxylation, suggesting inhibition of CYP3A catalytic activity. Meclizine has also been reported to be an inverse agonist and antagonist of human constitutive androstane receptor (hCAR). Therefore, given that certain tissues (e.g., liver) express both hPXR and hCAR and that various genes are cross-regulated by them, we quantified the expression of a hCAR- and hPXR-regulated gene (CYP2B6) in cultured human hepatocytes treated with meclizine. This drug did not decrease constitutive CYP2B6 mRNA expression or attenuate hCAR agonist-mediated increase in CYP2B6 mRNA and CYP2B6-catalyzed bupropion hydroxylation levels. These observations reflect hPXR agonism and the lack of hCAR inverse agonism and antagonism by meclizine, which were assessed by a hCAR reporter gene assay and mammalian two-hybrid assay. In conclusion, meclizine is a hPXR agonist, and it does not act as a hCAR inverse agonist or antagonist in cultured human hepatocytes. PMID:21131266

  9. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    PubMed

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. PMID:25449269

  10. Detecting constitutive activity and protean agonism at cannabinoid-2 receptor.

    PubMed

    Beltramo, Massimiliano; Brusa, Rossella; Mancini, Isabella; Scandroglio, Paola

    2010-01-01

    Since the cannabinoid system is involved in regulating several physiological functions such as locomotor activity, cognition, nociception, food intake, and inflammatory reaction, it has been the subject of intense study. Research on the pharmacology of this system has enormously progressed in the last 20years. One intriguing aspect that emerged from this research is that cannabinoid receptors (CBs) express a high level of constitutive activity. Investigation on this particular aspect of receptor pharmacology has largely focused on CB1, the CB subtype highly expressed in several brain regions. More recently, research on constitutive activity on the other CB subtype, CB2, was stimulated by the increasing interest on its potential as target for the treatment of various pathologies (e.g., pain and inflammation). There are several possible implications of constitutive activity on the therapeutic action of both agonists and antagonists, and consequently, it is important to have valuable methods to study this aspect of CB2 pharmacology. In the present chapter, we describe three methods to study constitutive activity at CB2: two classical methods relying on the detection of changes in cAMP level and GTPγS binding and a new one based on cell impedance measurement. In addition, we also included a section on detection of protean agonism, which is an interesting pharmacological phenomenon strictly linked to constitutive activity. PMID:21036225

  11. Bioluminescence imaging of estrogen receptor activity during breast cancer progression

    PubMed Central

    Vantaggiato, Cristina; Dell’Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation. PMID:27069764

  12. Propagation of conformational changes during μ-opioid receptor activation

    PubMed Central

    Sounier, Rémy; Mas, Camille; Steyaert, Jan; Laeremans, Toon; Manglik, Aashish; Huang, Weijiao; Kobilka, Brian; Déméné, Héléne; Granier, Sébastien

    2016-01-01

    μ-Opioid receptors (μOR) are G protein coupled receptors (GPCRs) that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive1 and agonist-induced active states (companion article) provide snapshots of the receptor at the beginning and end of a signaling event, but little is known about the dynamic sequence of events that span these two states. Here we report the use of solution-state NMR to examine the process of μOR activation. We obtained spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU72 alone, or with BU72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments (TM) 5 and 6, which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody revealing a weak allosteric coupling between the agonist binding pocket and the G protein coupling interface (TM5 and TM6) similar to what has been observed for the β2-adrenergic receptor2. Unexpectedly, in the presence of agonist alone, we observe larger spectral changes involving intracellular loop 1 (ICL1) and helix 8 (H8), when compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and ICL1 and/or H8 may play a role in G protein coupling specificity as has been suggested for other family A GPCRs. PMID:26245377

  13. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    PubMed

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation. PMID:27069764

  14. Cleavage and activation of a Toll-like receptor by microbial proteases

    PubMed Central

    de Zoete, Marcel R.; Bouwman, Lieneke I.; Keestra, A. Marijke; van Putten, Jos P. M.

    2011-01-01

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB–dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  15. Cleavage and activation of a Toll-like receptor by microbial proteases.

    PubMed

    de Zoete, Marcel R; Bouwman, Lieneke I; Keestra, A Marijke; van Putten, Jos P M

    2011-03-22

    Toll-like receptors (TLRs) are innate receptors that show high conservation throughout the animal kingdom. Most TLRs can be clustered into phylogenetic groups that respond to similar types of ligands. One exception is avian TLR15. This receptor does not categorize into one of the existing groups of TLRs and its ligand is still unknown. Here we report that TLR15 is a sensor for secreted virulence-associated fungal and bacterial proteases. Activation of TLR15 involves proteolytic cleavage of the receptor ectodomain and stimulation of NF-κB-dependent gene transcription. Receptor activation can be mimicked by the expression of a truncated TLR15 of which the entire ectodomain is removed, suggesting that receptor cleavage alleviates receptor inhibition by the leucine-rich repeat domain. Our results indicate TLR15 as a unique type of innate immune receptor that combines TLR characteristics with an activation mechanism typical for the evolutionary distinct protease-activated receptors. PMID:21383168

  16. Activation of glycine receptors modulates spontaneous epileptiform activity in the immature rat hippocampus

    PubMed Central

    Chen, Rongqing; Okabe, Akihito; Sun, Haiyan; Sharopov, Salim; Hanganu-Opatz, Ileana L; Kolbaev, Sergei N; Fukuda, Atsuo; Luhmann, Heiko J; Kilb, Werner

    2014-01-01

    While the expression of glycine receptors in the immature hippocampus has been shown, no information about the role of glycine receptors in controlling the excitability in the immature CNS is available. Therefore, we examined the effect of glycinergic agonists and antagonists in the CA3 region of an intact corticohippocampal preparation of the immature (postnatal days 4–7) rat using field potential recordings. Bath application of 100 μm taurine or 10 μm glycine enhanced the occurrence of recurrent epileptiform activity induced by 20 μm 4-aminopyridine in low Mg2+ solution. This proconvulsive effect was prevented by 3 μm strychnine or after incubation with the loop diuretic bumetanide (10 μm), suggesting that it required glycine receptors and an active NKCC1-dependent Cl− accumulation. Application of higher doses of taurine (≥1 mm) or glycine (100 μm) attenuated recurrent epileptiform discharges. The anticonvulsive effect of taurine was also observed in the presence of the GABAA receptor antagonist gabazine and was attenuated by strychnine, suggesting that it was partially mediated by glycine receptors. Bath application of the glycinergic antagonist strychnine (0.3 μm) induced epileptiform discharges. We conclude from these results that in the immature hippocampus, activation of glycine receptors can mediate both pro- and anticonvulsive effects, but that a persistent activation of glycine receptors is required to suppress epileptiform activity. In summary, our study elucidated the important role of glycine receptors in the control of neuronal excitability in the immature hippocampus. PMID:24665103

  17. Allergens and Activation of the Toll-Like Receptor Response.

    PubMed

    Monie, Tom P; Bryant, Clare E

    2016-01-01

    Pattern recognition receptors (PRRs) provide a crucial function in the detection of exogenous and endogenous danger signals. The Toll-like receptors (TLRs) were the first family of PRRs to be discovered and have been extensively studied since. Whilst TLRs remain the best characterized family of PRRs there is still much to be learnt about their mode of activation and the mechanisms of signal transduction they employ. Much of our understanding of these processes has been gathered through the use of cell based signaling assays utilizing specific gene-reporters or cytokine secretion based readouts. More recently it has become apparent that the repertoire of ligands recognized by these receptors may be wider than originally assumed and that their activation may be sensitized, or at least modulated by the presence of common household allergens such as the cat dander protein Fel d 1, or the house dust mite allergen Der p 2. In this chapter we provide an overview of the cell culture and stimulation processes required to study TLR signaling in HEK293 based assays and in bone marrow-derived macrophages. PMID:26803639

  18. Structural rearrangement of the intracellular domains during AMPA receptor activation.

    PubMed

    Zachariassen, Linda G; Katchan, Ljudmila; Jensen, Anna G; Pickering, Darryl S; Plested, Andrew J R; Kristensen, Anders S

    2016-07-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact. PMID:27313205

  19. Identification of Modulators of the Nuclear Receptor Peroxisome Proliferator-Activated Receptor α (PPARα) in a Mouse Liver Gene Expression Compendium

    EPA Science Inventory

    The nuclear receptor family member peroxisome proliferator-activated receptor α (PPARα) is activated by therapeutic hypolipidemic drugs and environmentally-relevant chemicals to regulate genes involved in lipid transport and catabolism. Chronic activation of PPARα in rodents inc...

  20. Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

    PubMed Central

    Abdulkhalek, Samar

    2010-01-01

    Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed 1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB 3, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors 4. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 5. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a

  1. D1 dopamine receptor activity of anti-parkinsonian drugs.

    PubMed

    Fici, G J; Wu, H; VonVoigtlander, P F; Sethy, V H

    1997-01-01

    Clinical and preclinical investigations suggest that stimulation of D1 dopamine receptors may be responsible for dyskinesias induced by dopamine agonist treatment of Parkinson's Disease (PD), and that these dyskinesias may be decreased by treatment with a D1 antagonist (clozapine). Therefore, the effects of dopamine agonists and antagonists have been investigated in a primary cerebellar granule cell model of cAMP formation that seems to be highly responsive to the D1 receptors. SKF 38393, lisuride, apomorphine, pergolide, dopamine, bromocriptine and 7-OH-DPAT showed concentration-dependent increases in cAMP formation, with EC50s (in microM) of 0.013, 0.053, 0.25, 1.04, 2.18, 50.9 and 54.4, respectively. SKF 38393, apomorphine, dopamine and pergolide had similar intrinsic activity (100%), while the intrinsic activities of 7-OH-DPAT, bromocriptine and lisuride were 28.0%, 20.7% and 17.2%, respectively. SCH 23390, a selective D1 dopamine receptor antagonist, blocked an increase in cAMP formation produced by EC50 concentrations of all of the dopamine agonists investigated in this study. Clozapine concentration-dependently blocked pergolide-induced increases in cAMP and was approximately 1700-fold less potent than SCH 23390 (IC50: 0.97 microM and 0.56 nM, respectively). U-95666A (1-1000 microM), selective for the D2 receptors, showed no significant effect on cAMP, while pramipexole (0.1-100 microM), a D3 preferring agonist, did not elevate cAMP. These data suggest that primary cerebellar granule cell cultures are an excellent model for measuring D1 dopamine receptor-mediated changes in cellular cAMP. The results are discussed with reference to the relationship between the D1 receptor-stimulated increase in cAMP formation and the induction of dyskinesia in humans by these anti-parkinsonian drugs. PMID:9126882

  2. Vortioxetine, but not escitalopram or duloxetine, reverses memory impairment induced by central 5-HT depletion in rats: evidence for direct 5-HT receptor modulation.

    PubMed

    Jensen, Jesper Bornø; du Jardin, Kristian Gaarn; Song, Dekun; Budac, David; Smagin, Gennady; Sanchez, Connie; Pehrson, Alan Lars

    2014-01-01

    Depressed patients suffer from cognitive dysfunction, including memory deficits. Acute serotonin (5-HT) depletion impairs memory and mood in vulnerable patients. The investigational multimodal acting antidepressant vortioxetine is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, 5-HT1B receptor partial agonist, 5-HT1A receptor agonist and 5-HT transporter (SERT) inhibitor that enhances memory in normal rats in novel object recognition (NOR) and conditioned fear (Mørk et al., 2013). We hypothesized that vortioxetine's 5-HT receptor mechanisms are involved in its memory effects, and therefore investigated these effects in 5-HT depleted rats. Four injections of the irreversible tryptophan hydroxylase inhibitor 4-chloro-dl-phenylalanine methyl ester hydrochloride (PCPA, 86mg/kg, s.c.) induced 5-HT depletion, as measured in hippocampal homogenate and microdialysate. The effects of acute challenge with vortioxetine or the 5-HT releaser fenfluramine on extracellular 5-HT were measured in PCPA-treated and control rats. PCPA's effects on NOR and spontaneous alternation (SA) performance were assessed along with the effects of acute treatment with 5-hydroxy-l-tryptophan (5-HTP), vortioxetine, the selective 5-HT reuptake inhibitor escitalopram, or the 5-HT norepinephrine reuptake inhibitor duloxetine. SERT occupancies were estimated by ex vivo autoradiography. PCPA depleted central 5-HT by >90% in tissue and microdialysate, and impaired NOR and SA performance. Restoring central 5-HT with 5-HTP reversed these deficits. At similar SERT occupancies (>90%) vortioxetine, but not escitalopram or duloxetine, restored memory performance. Acute fenfluramine significantly increased extracellular 5-HT in control and PCPA-treated rats, while vortioxetine did so only in control rats. Thus, vortioxetine restores 5-HT depletion impaired memory performance in rats through one or more of its receptor activities. PMID:24284262

  3. Estrogen receptor profiling and activity in cardiac myocytes.

    PubMed

    Pugach, Emily K; Blenck, Christa L; Dragavon, Joseph M; Langer, Stephen J; Leinwand, Leslie A

    2016-08-15

    Estrogen signaling appears critical in the heart. However a mechanistic understanding of the role of estrogen in the cardiac myocyte is lacking. Moreover, there are multiple cell types in the heart and multiple estrogen receptor (ER) isoforms. Therefore, we studied expression, localization, transcriptional and signaling activity of ERs in isolated cardiac myocytes. We found only ERα RNA (but no ERβ RNA) in cardiac myocytes using two independent methods. The vast majority of full-length ERα protein (ERα66) localizes to cardiac myocyte nuclei where it is competent to activate transcription. Alternate isoforms of ERα encoded by the same genomic locus (ERα46 and ERα36) have differential transcriptional activity in cardiac myocytes but also primarily localize to nuclei. In contrast to other reports, no ERα isoform is competent to activate MAPK or PI3K signaling in cardiac myocytes. Together these data support a role for ERα at the level of transcription in cardiac myocytes. PMID:27164442

  4. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    PubMed Central

    Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

    2012-01-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  5. The Pharmacochaperone Activity of Quinine on Bitter Taste Receptors.

    PubMed

    Upadhyaya, Jasbir D; Chakraborty, Raja; Shaik, Feroz A; Jaggupilli, Appalaraju; Bhullar, Rajinder P; Chelikani, Prashen

    2016-01-01

    Bitter taste is one of the five basic taste sensations which is mediated by 25 bitter taste receptors (T2Rs) in humans. The mechanism of bitter taste signal transduction is not yet elucidated. The cellular processes underlying T2R desensitization including receptor internalization, trafficking and degradation are yet to be studied. Here, using a combination of molecular and pharmacological techniques we show that T2R4 is not internalized upon agonist treatment. Pretreatment with bitter agonist quinine led to a reduction in subsequent quinine-mediated calcium responses to 35 ± 5% compared to the control untreated cells. Interestingly, treatment with different bitter agonists did not cause internalization of T2R4. Instead, quinine treatment led to a 2-fold increase in T2R4 cell surface expression which was sensitive to Brefeldin A, suggesting a novel pharmacochaperone activity of quinine. This phenomenon of chaperone activity of quinine was also observed for T2R7, T2R10, T2R39 and T2R46. Our results suggest that the observed action of quinine for these T2Rs is independent of its agonist activity. This study provides novel insights into the pharmacochaperone activity of quinine and possible mechanism of T2R desensitization, which is of fundamental importance in understanding the mechanism of bitter taste signal transduction. PMID:27223611

  6. Helix 11 dynamics is critical for constitutive androstane receptor activity.

    PubMed

    Wright, Edward; Busby, Scott A; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R; Fernandez, Elias J

    2011-01-12

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Hydrogen/deuterium exchange (HDX) data indicate that the CAR activation function 2 (AF-2) is more stable in CAR(TCPOBOP):RXR and CAR(meclizine):RXR than in CAR:RXR. HDX kinetics also show significant differences between CAR(TCPOBOP):RXR and CAR(meclizine):RXR. Unlike CAR(meclizine):RXR, CAR(TCPOBOP):RXR shows a higher overall stabilization that extends into RXR. We identify residues 339-345 in CAR as an allosteric regulatory site with a greater magnitude reduction in exchange kinetics in CAR(TCPOBOP):RXR than CAR(meclizine):RXR. Accordingly, assays with mutations on CAR at leucine-340 and leucine-343 confirm this region as an important determinant of CAR activity. PMID:21220114

  7. The Pharmacochaperone Activity of Quinine on Bitter Taste Receptors

    PubMed Central

    Upadhyaya, Jasbir D.; Chakraborty, Raja; Shaik, Feroz A.; Jaggupilli, Appalaraju; Bhullar, Rajinder P.; Chelikani, Prashen

    2016-01-01

    Bitter taste is one of the five basic taste sensations which is mediated by 25 bitter taste receptors (T2Rs) in humans. The mechanism of bitter taste signal transduction is not yet elucidated. The cellular processes underlying T2R desensitization including receptor internalization, trafficking and degradation are yet to be studied. Here, using a combination of molecular and pharmacological techniques we show that T2R4 is not internalized upon agonist treatment. Pretreatment with bitter agonist quinine led to a reduction in subsequent quinine-mediated calcium responses to 35 ± 5% compared to the control untreated cells. Interestingly, treatment with different bitter agonists did not cause internalization of T2R4. Instead, quinine treatment led to a 2-fold increase in T2R4 cell surface expression which was sensitive to Brefeldin A, suggesting a novel pharmacochaperone activity of quinine. This phenomenon of chaperone activity of quinine was also observed for T2R7, T2R10, T2R39 and T2R46. Our results suggest that the observed action of quinine for these T2Rs is independent of its agonist activity. This study provides novel insights into the pharmacochaperone activity of quinine and possible mechanism of T2R desensitization, which is of fundamental importance in understanding the mechanism of bitter taste signal transduction. PMID:27223611

  8. DHEA metabolites activate estrogen receptors alpha and beta

    PubMed Central

    Michael Miller, Kristy K.; Al-Rayyan, Numan; Ivanova, Margarita M.; Mattingly, Kathleen A.; Ripp, Sharon L.; Klinge, Carolyn M.; Prough, Russell A.

    2012-01-01

    Dehydroepiandrosterone (DHEA) levels were reported to associate with increased breast cancer risk in postmenopausal women, but some carcinogen-induced rat mammary tumor studies question this claim. The purpose of this study was to determine how DHEA and its metabolites affect estrogen receptors α or β (ERα or ERβ) -regulated gene transcription and cell proliferation. In transiently transfected HEK-293 cells, androstenediol, DHEA, and DHEA-S activated ERα. In ERβ transfected HepG2 cells, androstenedione, DHEA, androstenediol, and 7-oxo DHEA stimulated reporter activity. ER antagonists ICI 182,780 (fulvestrant) and 4-hydroxytamoxifen, general P450 inhibitor miconazole, and aromatase inhibitor exemestane inhibited activation by DHEA or metabolites in transfected cells. ERβ-selective antagonist R,R-THC (R,R-cis-diethyl tetrahydrochrysene) inhibited DHEA and DHEA metabolite transcriptional activity in ERβ-transfected cells. Expression of endogenous estrogen-regulated genes: pS2, progesterone receptor, cathepsin D1, and nuclear respiratory factor-1 was increased by DHEA and its metabolites in an ER-subtype, gene, and cell-specific manner. DHEA metabolites, but not DHEA, competed with 17β-estradiol for ERα and ERβ binding and stimulated MCF-7 cell proliferation, demonstrating that DHEA metabolites interact directly with ERα and ERβ in vitro, modulating estrogen target genes in vivo. PMID:23123738

  9. Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3

    PubMed Central

    Burnier, Laurent

    2013-01-01

    The direct cytoprotective activities of activated protein C (APC) on cells convey therapeutic, relevant, beneficial effects in injury and disease models in vivo and require the endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1). Thrombin also activates PAR1, but its effects on cells contrast APC’s cytoprotective effects. To gain insights into mechanisms for these contrasting cellular effects, protease activated receptor 3 (PAR3) activation by APC and thrombin was studied. APC cleaved PAR3 on transfected and endothelial cells in the presence of EPCR. Remarkably, APC cleaved a synthetic PAR3 N-terminal peptide at Arg41, whereas thrombin cleaved at Lys38. On cells, APC failed to cleave R41Q-PAR3, whereas K38Q-PAR3 was still cleaved by APC but not by thrombin. PAR3 tethered-ligand peptides beginning at amino acid 42, but not those beginning at amino acid 39, conveyed endothelial barrier-protective effects. In vivo, the APC-derived PAR3 tethered-ligand peptide, but not the thrombin-derived PAR3 peptide, blunted vascular endothelial growth factor (VEGF)-induced vascular permeability. These data indicate that PAR3 cleavage by APC at Arg41 can initiate distinctive APC-like cytoprotective effects. These novel insights help explain the differentiation of APC’s cytoprotective versus thrombin’s proinflammatory effects on cells and suggest a unique contributory role for PAR3 in the complex mechanisms underlying APC cytoprotective effects. PMID:23788139

  10. Activation of type 5 metabotropic glutamate receptors attenuates deficits in cognitive flexibility induced by NMDA receptor blockade

    PubMed Central

    Stefani, Mark R.; Moghaddam, Bita

    2010-01-01

    Metabotropic glutamate (mGlu) receptors provide a mechanism by which the function of NMDA glutamate receptors can be modulated. As NMDA receptor hypofunction is implicated in the etiology of psychiatric disorders, including schizophrenia, the pharmacological regulation of mGlu receptor activity represents a promising therapeutic approach. We examined the effects of the positive allosteric mGlu5 receptor modulator 3- cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), alone and in combination with the NMDA receptor antagonist MK-801, on a task measuring cognitive set-shifting ability. This task measures NMDA receptor-dependent cognitive abilities analogous to those impaired in schizophrenia. Systemic administration of CDPPB (10 & 30 mg/kg i.p) blocked MK-801 (0.1 mg/kg, i.p.)-induced impairments in set-shifting ability. The effect on learning was dose-dependent, with the 30 mg/kg dose having a greater effect than the 10 mg/kg dose across all trials. This ameliorative effect of CDPPB reflected a reduction in MK-801-induced perseverative responding. These results add to the evidence that mGlu5 receptors interact functionally with NMDA receptors to regulate behavior, and suggest that positive modulators of mGlu5 receptors may have therapeutic potential in the treatment of disorders, like schizophrenia, characterized by impairments in cognitive flexibility and memory. PMID:20371234

  11. Synaptic activity regulates AMPA receptor trafficking through different recycling pathways.

    PubMed

    Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

    2015-01-01

    Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. PMID:25970033

  12. Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

    PubMed Central

    Gaskill, Peter J.; Yano, Hideaki H.; Kalpana, Ganjam V.; Javitch, Jonathan A.; Berman, Joan W.

    2014-01-01

    Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers. PMID:25268786

  13. Activation of family C G-protein-coupled receptors by the tripeptide glutathione.

    PubMed

    Wang, Minghua; Yao, Yi; Kuang, Donghui; Hampson, David R

    2006-03-31

    The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the gamma-aminobutyric acid, type B (GABAB) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acid-activated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or co-ligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [3H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wild-type and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABAB receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions. PMID:16455645

  14. Aryl hydrocarbon receptor-independent activation of estrogen receptor-dependent transcription by 3-methycholanthrene

    SciTech Connect

    Shipley, Jonathan M.; Waxman, David J. . E-mail: djw@bu.edu

    2006-06-01

    Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that stimulates transcription directed by xenobiotic response elements upstream of target genes. Recently, AhR ligands were reported to induce formation of an AhR-estrogen receptor (ER) complex, which can bind to estrogen response elements (EREs) and stimulate transcription of ER target genes. Presently, we investigate the effect of the AhR ligands 3-methylcholanthrene (3MC), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (BZ126) on ERE-regulated luciferase reporter activity and endogenous ER target gene expression. In MCF-7 human breast cancer cells, 3MC induced transcription of ER reporter genes containing native promoter sequences of the ER-responsive genes complement 3 and pS2 and heterologous promoters regulated by isolated EREs. Dose-response studies revealed that the concentration of 3MC required to half-maximally activate transcription (EC{sub 5}) was >100-fold higher for an ER reporter (27-57 {mu}M) than for an AhR reporter (86-250 nM) in both MCF-7 cells and in human endometrial cancer Ishikawa cells. 3MC also stimulated expression of the endogenous ER target genes amphiregulin, cathepsin D and progesterone receptor, albeit to a much lower extent than was achieved following stimulation with 17{beta}-estradiol. In Ishikawa cells, 3MC, but not BZ126 or TCDD, stimulated ER{alpha}-dependent reporter activity but did not induce expression of endogenous ER target genes. Finally, studies carried out in the AhR-positive rat hepatoma cell line 5L and the AhR-deficient variant BP8 demonstrated that ER reporter activity could be induced by 3MC in a manner that was independent of AhR and thus distinct from the AhR-ER 'hijacking' mechanism described recently. 3MC may thus elicit estrogenic activity by multiple mechanisms.

  15. Involvement of spinal muscarinic and serotonergic receptors in the anti-allodynic effect of electroacupuncture in rats with oxaliplatin-induced neuropathic pain

    PubMed Central

    Lee, Ji Hwan; Go, Donghyun; Kim, Woojin; Lee, Giseog; Bae, Hyojeong; Quan, Fu Shi

    2016-01-01

    This study was performed to investigate whether the spinal cholinergic and serotonergic analgesic systems mediate the relieving effect of electroacupuncture (EA) on oxaliplatin-induced neuropathic cold allodynia in rats. The cold allodynia induced by an oxaliplatin injection (6 mg/kg, i.p.) was evaluated by immersing the rat's tail into cold water (4℃) and measuring the withdrawal latency. EA stimulation (2 Hz, 0.3-ms pulse duration, 0.2~0.3 mA) at the acupoint ST36, GV3, or LI11 all showed a significant anti-allodynic effect, which was stronger at ST36. The analgesic effect of EA at ST36 was blocked by intraperitoneal injection of muscarinic acetylcholine receptor antagonist (atropine, 1 mg/kg), but not by nicotinic (mecamylamine, 2 mg/kg) receptor antagonist. Furthermore, intrathecal administration of M2 (methoctramine, 10 µg) and M3 (4-DAMP, 10 µg) receptor antagonist, but not M1 (pirenzepine, 10 µg) receptor antagonist, blocked the effect. Also, spinal administration of 5-HT3 (MDL-72222, 12 µg) receptor antagonist, but not 5-HT1A (NAN-190, 15 µg) or 5-HT2A (ketanserin, 30 µg) receptor antagonist, prevented the anti-allodynic effect of EA. These results suggest that EA may have a signifi cant analgesic action against oxaliplatin-induced neuropathic pain, which is mediated by spinal cholinergic (M2, M3) and serotonergic (5-HT3) receptors. PMID:27382357

  16. Structural activation pathways from dynamic olfactory receptor-odorant interactions.

    PubMed

    Lai, Peter C; Singer, Michael S; Crasto, Chiquito J

    2005-11-01

    We have simulated an odor ligand's dynamic behavior in the binding region of an olfactory receptor (OR). Our short timescale computational studies (up to 200 ps) have helped identify unprecedented postdocking ligand behavior of ligands. From in vacuo molecular dynamics simulations of interactions between models of rat OR I7 and 10 aldehyde ligands, we have identified a dissociative pathway along which the ligand exits and enters the OR-binding pocket--a transit event. The ligand's transit through the receptor's binding region may mark the beginning of a signal transduction cascade leading to odor recognition. We have graphically traced the rotameric changes in key OR amino acid side chains during the transit. Our results have helped substantiate or refute previously held notions of amino acid contribution to ligand stability in the binding pocket. Our observations of ligand activity when compared to those of experimental (electroolfactogram response) OR-activation studies provide a view to predicting the stability of ligands in the binding pocket as a precursor to OR activation by the ligand. PMID:16243965

  17. Peroxisome proliferator-activated receptor {alpha}-independent peroxisome proliferation

    SciTech Connect

    Zhang Xiuguo; Tanaka, Naoki . E-mail: naopi@hsp.md.shinshu-u.ac.jp; Nakajima, Takero; Kamijo, Yuji; Gonzalez, Frank J.; Aoyama, Toshifumi

    2006-08-11

    Hepatic peroxisome proliferation, increases in the numerical and volume density of peroxisomes, is believed to be closely related to peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}) activation; however, it remains unknown whether peroxisome proliferation depends absolutely on this activation. To verify occurrence of PPAR{alpha}-independent peroxisome proliferation, fenofibrate treatment was used, which was expected to significantly enhance PPAR{alpha} dependence in the assay system. Surprisingly, a novel type of PPAR{alpha}-independent peroxisome proliferation and enlargement was uncovered in PPAR{alpha}-null mice. The increased expression of dynamin-like protein 1, but not peroxisome biogenesis factor 11{alpha}, might be associated with the PPAR{alpha}-independent peroxisome proliferation at least in part.

  18. Kainate receptor activation induces glycine receptor endocytosis through PKC deSUMOylation.

    PubMed

    Sun, Hao; Lu, Li; Zuo, Yong; Wang, Yan; Jiao, Yingfu; Zeng, Wei-Zheng; Huang, Chao; Zhu, Michael X; Zamponi, Gerald W; Zhou, Tong; Xu, Tian-Le; Cheng, Jinke; Li, Yong

    2014-01-01

    Surface expression and regulated endocytosis of glycine receptors (GlyRs) play a critical function in balancing neuronal excitability. SUMOylation (SUMO modification) is of critical importance for maintaining neuronal function in the central nervous system. Here we show that activation of kainate receptors (KARs) causes GlyR endocytosis in a calcium- and protein kinase C (PKC)-dependent manner, leading to reduced GlyR-mediated synaptic activity in cultured spinal cord neurons and the superficial dorsal horn of rat spinal cord slices. This effect requires SUMO1/sentrin-specific peptidase 1 (SENP1)-mediated deSUMOylation of PKC, indicating that the crosstalk between KARs and GlyRs relies on the SUMOylation status of PKC. SENP1-mediated deSUMOylation of PKC is involved in the kainate-induced GlyR endocytosis and thus plays an important role in the anti-homeostatic regulation between excitatory and inhibitory ligand-gated ion channels. Altogether, we have identified a SUMOylation-dependent regulatory pathway for GlyR endocytosis, which may have important physiological implications for proper neuronal excitability. PMID:25236484

  19. Artificial masculinization in tilapia involves androgen receptor activation.

    PubMed

    Golan, Matan; Levavi-Sivan, Berta

    2014-10-01

    Estrogens have a pivotal role in natural female sexual differentiation of tilapia while lack of steroids results in testicular development. Despite the fact that androgens do not participate in natural sex differentiation, synthetic androgens, mainly 17-α-methyltestosterone (MT) are effective in the production of all-male fish in aquaculture. The sex inversion potency of synthetic androgens may arise from their androgenic activity or else as inhibitors of aromatase activity. The current study is an attempt to differentiate between the two alleged activities in order to evaluate their contribution to the sex inversion process and aid the search for novel sex inversion agents. In the present study, MT inhibited aromatase activity, when applied in vitro as did the non-aromatizable androgen dihydrotestosterone (DHT). In comparison, exposure to fadrozole, a specific aromatase inhibitor, was considerably more effective. Androgenic activity of MT was evaluated by exposure of Sciaenochromis fryeri fry to the substance and testing for the appearance of blue color. Flutamide, an androgen antagonist, administered concomitantly with MT, reduced the appearance of the blue color and the sex inversion potency of MT in a dose-dependent manner. In tilapia, administration of MT, fadrozole or DHT resulted in efficient sex inversion while flutamide reduced the sex inversion potency of all three compounds. In the case of MT and DHT the decrease in sex inversion efficiency caused by flutamide is most likely due to the direct blocking of the androgen binding to its cognate receptor. The negative effect of flutamide on the efficiency of the fadrozole treatment may indicate that the masculinizing activity of fadrozole may be attributed to excess, un-aromatized, androgens accumulated in the differentiating gonad. The present study shows that when androgen receptors are blocked, there is a reduction in the efficiency of sex inversion treatments. Our results suggest that in contrast to

  20. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

    PubMed Central

    Santulli, R J; Derian, C K; Darrow, A L; Tomko, K A; Eckardt, A J; Seiberg, M; Scarborough, R M; Andrade-Gordon, P

    1995-01-01

    Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2 [Nystedt, S., Emilsson, K., Wahlestedt, C. & Sundelin, J. (1994) Proc. Natl. Acad. Sci. USA 91, 9208-9212]. PAR-2 is activated by nanomolar concentrations of trypsin and possesses the tethered ligand sequence SLIGRL. SLIGRL was found to be equipotent with SFLLRN in activating keratinocyte inositolphospholipid hydrolysis and calcium mobilization. Desensitization studies indicated that SFLLRN, SLIGRL, and trypsin activate a common receptor, PAR-2. Northern blot analyses detected a transcript of PAR-2 in total RNA from keratinocytes but not fibroblasts. Levels of thrombin receptor message were equivalent in the two cell types. Our results indicate that human keratinocytes possess PAR-2, suggesting a potential role for this receptor in tissue repair and/or skin-related disorders. Images Fig. 6 PMID:7568091

  1. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    SciTech Connect

    Onuma, Hirohisa; Inukai, Kouichi Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  2. The Role of Hippocampal NMDA Receptors in Long-Term Emotional Responses following Muscarinic Receptor Activation

    PubMed Central

    Hoeller, Alexandre A.; Costa, Ana Paula R.; Bicca, Maíra A.; Matheus, Filipe C.; Lach, Gilliard; Spiga, Francesca; Lightman, Stafford L.; Walz, Roger; Collingridge, Graham L.; Bortolotto, Zuner A.; de Lima, Thereza C. M.

    2016-01-01

    Extensive evidence indicates the influence of the cholinergic system on emotional processing. Previous findings provided new insights into the underlying mechanisms of long-term anxiety, showing that rats injected with a single systemic dose of pilocarpine—a muscarinic receptor (mAChR) agonist—displayed persistent anxiogenic-like responses when evaluated in different behavioral tests and time-points (24 h up to 3 months later). Herein, we investigated whether the pilocarpine-induced long-term anxiogenesis modulates the HPA axis function and the putative involvement of NMDA receptors (NMDARs) following mAChRs activation. Accordingly, adult male Wistar rats presented anxiogenic-like behavior in the elevated plus-maze (EPM) after 24 h or 1 month of pilocarpine injection (150 mg/kg, i.p.). In these animals, mAChR activation disrupted HPA axis function inducing a long-term increase of corticosterone release associated with a reduced expression of hippocampal GRs, as well as consistently decreased NMDAR subunits expression. Furthermore, in another group of rats injected with memantine–an NMDARs antagonist (4 mg/kg, i.p.)–prior to pilocarpine, we found inhibition of anxiogenic-like behaviors in the EPM but no further alterations in the pilocarpine-induced NMDARs downregulation. Our data provide evidence that behavioral anxiogenesis induced by mAChR activation effectively yields short- and long-term alterations in hippocampal NMDARs expression associated with impairment of hippocampal inhibitory regulation of HPA axis activity. This is a novel mechanism associated with anxiety-like responses in rats, which comprise a putative target to future translational studies. PMID:26795565

  3. Mode of action framework analysis for receptor-mediated toxicity: the Peroxisome Proliferator-Activated Receptor alpha (PPARα) as a case study

    EPA Science Inventory

    Therapeutic hypolipidemic agents and industrial chemicals that cause peroxisome proliferation and induce liver tumors in rodents activate the nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα). Research has elucidated the cellular and molecular events by w...

  4. Evolution of the protease-activated receptor family in vertebrates

    PubMed Central

    JIN, MIN; YANG, HAI-WEI; TAO, AI-LIN; WEI, JI-FU

    2016-01-01

    Belonging to the G protein-coupled receptor (GPcr) family, the protease-activated receptors (Pars) consist of 4 members, PAR1-4. PARs mediate the activation of cells via thrombin, serine and other proteases. Such protease-triggered signaling events are thought to be critical for hemostasis, thrombosis and other normal pathological processes. In the present study, we examined the evolution of PARs by analyzing phylogenetic trees, chromosome location, selective pressure and functional divergence based on the 169 functional gene alignment sequences from 57 vertebrate gene sequences. We found that the 4 PARs originated from 4 invertebrate ancestors by phylogenetic trees analysis. The selective pressure results revealed that only PAR1 appeared by positive selection during its evolution, while the other PAR members did not. In addition, we noticed that although these PARs evolved separately, the results of functional divergence indicated that their evolutional rates were similar and their functions did not significantly diverge. The findings of our study provide valuable insight into the evolutionary history of the vertebrate PAR family. PMID:26820116

  5. Antitussive activity of Withania somnifera and opioid receptors.

    PubMed

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways. PMID:25252908

  6. Peroxisome proliferator activated receptor-γ and traumatic brain injury

    PubMed Central

    Qi, Lei; Jacob, Asha; Wang, Ping; Wu, Rongqian

    2010-01-01

    Traumatic brain injury (TBI) represents a major health care problem and a significant socioeconomic challenge worldwide. No specific therapy for TBI is available. The peroxisome proliferator activated receptor-γ (PPAR-γ) belongs to the nuclear receptor superfamily. Although PPAR-γ was originally characterized in adipose tissue as a regulator of lipid and glucose metabolism, recent studies showed that PPAR-γ is present in most cell types and plays a central role in the regulation of adipogenesis, glucose homeostasis, cellular differentiation, apoptosis and inflammation. Here, we reviewed the current literature on the molecular mechanisms of PPAR-γ-related neuroprotection after TBI. Growing evidence has indicated that the beneficial effects of PPAR-γ activation in TBI appear to be mediated through downregulation of inflammatory responses, reduction of oxidative stress, inhibition of apoptosis, and promotion of neurogenesis. A thorough understanding of the PPAR-γ pathway will be critical to the development of therapeutic interventions for the treatment of patients with TBI. PMID:21072262

  7. Potentiation of glucocorticoid receptor transcriptional activity by sumoylation.

    PubMed

    Le Drean, Yves; Mincheneau, Nathalie; Le Goff, Pascale; Michel, Denis

    2002-09-01

    The glucocorticoid receptor (GR) is a transcription factor, subject to several types of posttranslational modifications including phosphorylation and ubiquitination. We showed that the GR is covalently modified by the small ubiquitin-related modifier-1 (SUMO-1) peptide in mammalian cells. We demonstrated that GR sumoylation is not dependent on the presence of the ligand and regulates the stability of the protein as well as its transcriptional activity. SUMO-1 overexpression induces dramatic GR degradation, abolished by proteasome inhibition. We also found that SUMO-1 stimulates the transactivation capacity of GRs to an extent largely exceeding those observed so far for other sumoylated transcription factors. Overexpression of SUMO-1 specifically enhances the ligand-induced transactivation of GR up to 8-fold. However, this hyperactivation occurs only in the context of a synergy between multiple molecules of GRs. It requires more than one receptor DNA-binding site in promoter and becomes more prominent as the number of sites increases. Interestingly, these observations may be related to the transcriptional properties of the synergy control region of GRs, which precisely contains two evolutionary conserved sumoylation sites. We propose a model in which SUMO-1 regulates the synergy control function of GR and serves as a unique signal for activation and destruction. PMID:12193561

  8. In vitro neuronal network activity in NMDA receptor encephalitis

    PubMed Central

    2013-01-01

    Background Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR) and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. Results We describe effects of cerebrospinal fluid (CSF) from an anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis patient on in vitro neuronal network activity (ivNNA). In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA) system. The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF) taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. Conclusion Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes. PMID:23379293

  9. A restricted population of CB1 cannabinoid receptors with neuroprotective activity

    PubMed Central

    Chiarlone, Anna; Bellocchio, Luigi; Blázquez, Cristina; Resel, Eva; Soria-Gómez, Edgar; Cannich, Astrid; Ferrero, José J.; Sagredo, Onintza; Benito, Cristina; Romero, Julián; Sánchez-Prieto, José; Lutz, Beat; Fernández-Ruiz, Javier; Galve-Roperh, Ismael; Guzmán, Manuel

    2014-01-01

    The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. Of note, CB1 receptors are expressed at the synapses of two opposing (i.e., GABAergic/inhibitory and glutamatergic/excitatory) neuronal populations, so the activation of one and/or another receptor population may conceivably evoke different effects. Despite the widely reported neuroprotective activity of the CB1 receptor in animal models, the precise pathophysiological relevance of those two CB1 receptor pools in neurodegenerative processes is unknown. Here, we first induced excitotoxic damage in the mouse brain by (i) administering quinolinic acid to conditional mutant animals lacking CB1 receptors selectively in GABAergic or glutamatergic neurons, and (ii) manipulating corticostriatal glutamatergic projections remotely with a designer receptor exclusively activated by designer drug pharmacogenetic approach. We next examined the alterations that occur in the R6/2 mouse, a well-established model of Huntington disease, upon (i) fully knocking out CB1 receptors, and (ii) deleting CB1 receptors selectively in corticostriatal glutamatergic or striatal GABAergic neurons. The data unequivocally identify the restricted population of CB1 receptors located on glutamatergic terminals as an indispensable player in the neuroprotective activity of (endo)cannabinoids, therefore suggesting that this precise receptor pool constitutes a promising target for neuroprotective therapeutic strategies. PMID:24843137

  10. Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis.

    PubMed

    McEachron, Troy A; Pawlinski, Rafal; Richards, Kristy L; Church, Frank C; Mackman, Nigel

    2010-12-01

    The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis. PMID:20736455

  11. Protease-activated receptors mediate crosstalk between coagulation and fibrinolysis

    PubMed Central

    McEachron, Troy A.; Pawlinski, Rafal; Richards, Kristy L.; Church, Frank C.

    2010-01-01

    The coagulation and fibrinolytic systems contribute to malignancy by increasing angiogenesis, tumor growth, tumor invasion, and tumor metastasis. Oncogenic transformation increases the expression of tissue factor (TF) that results in local generation of coagulation proteases and activation of protease-activated receptor (PAR)-1 and PAR-2. We compared the PAR-dependent expression of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor (PAI)-1 in 2 murine mammary adencocarcinoma cell lines: metastatic 4T1 cells and nonmetastatic 67NR cells. 4T1 cells expressed TF, PAR-1 and PAR-2 whereas 67NR cells expressed TF and PAR-1. We also silenced PAR-1 or PAR-2 expression in the 4T1 cells. We discovered 2 distinct mechanisms for PAR-dependent expression of uPA and PAI-1. First, we found that factor Xa or thrombin activation of PAR-1 led to a rapid release of stored intracellular uPA into the culture supernatant. Second, thrombin transactivation of a PAR-1/PAR-2 complex resulted in increases in PAI-1 mRNA and protein expression. Cells lacking PAR-2 failed to express PAI-1 in response to thrombin and factor Xa did not activate the PAR-1/PAR-2 complex. Our results reveal how PAR-1 and PAR-2 on tumor cells mediate crosstalk between coagulation and fibrinolysis. PMID:20736455

  12. Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

    SciTech Connect

    Morrison, W.J.

    1988-01-01

    The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. ({sup 3}H)PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 {mu}M. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRP{gamma}S and GDP{beta}S, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA).

  13. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    PubMed

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability. PMID:22538662

  14. Thrombin stimulates insulin secretion via protease-activated receptor-3

    PubMed Central

    Hänzelmann, Sonja; Wang, Jinling; Güney, Emre; Tang, Yunzhao; Zhang, Enming; Axelsson, Annika S; Nenonen, Hannah; Salehi, Albert S; Wollheim, Claes B; Zetterberg, Eva; Berntorp, Erik; Costa, Ivan G; Castelo, Robert; Rosengren, Anders H

    2015-01-01

    The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes (‘module’) including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a ‘tethered ligand’ to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca2+ release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D. PMID:26742564

  15. Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor.

    PubMed

    Kota, Kokila; Kuzhikandathil, Eldo V; Afrasiabi, Milad; Lacy, Brett; Kontoyianni, Maria; Crider, A Michael; Song, Daniel

    2015-09-01

    The dopamine D3 receptor exhibits agonist-dependent tolerance and slow response termination (SRT) signaling properties that distinguish it from the closely-related D2 receptors. While amino acid residues important for D3 receptor ligand binding have been identified, the residues involved in activation of D3 receptor signaling and induction of signaling properties have not been determined. In this paper, we used cis and trans isomers of a novel D3 receptor agonist, 8-OH-PBZI, and site-directed mutagenesis to identify key residues involved in D3 receptor signaling function. Our results show that trans-8-OH-PBZI, but not cis-8-OH-PBZI, elicit the D3 receptor tolerance and SRT properties. We show that while both agonists require a subset of residues in the orthosteric binding site of D3 receptors for activation of the receptor, the ability of the two isomers to differentially induce tolerance and SRT is mediated by interactions with specific residues in the sixth transmembrane helix and third extracellular loop of the D3 receptor. We also show that unlike cis-8-OH-PBZI, which is a partial agonist at the dopamine D2S receptor and full agonist at dopamine D2L receptor, trans-8-OH-PBZI is a full agonist at both D2S and D2L receptors. The different effect of the two isomers on D3 receptor signaling properties and D2S receptor activation correlated with differential effects of the isomers on agonist-induced mouse locomotor activity. The two isomers of 8-OH-PBZI represent novel pharmacological tools for in silico D3 and D2 receptor homology modeling and for determining the role of D3 receptor tolerance and SRT properties in signaling and behavior. PMID:26116441

  16. Serotonin 5-HT2 Receptors Induce a Long-Lasting Facilitation of Spinal Reflexes Independent of Ionotropic Receptor Activity

    PubMed Central

    Shay, Barbara L.; Sawchuk, Michael; Machacek, David W.; Hochman, Shawn

    2009-01-01

    Dorsal root-evoked stimulation of sensory afferents in the hemisected in vitro rat spinal cord produces reflex output, recorded on the ventral roots. Transient spinal 5-HT2C receptor activation induces a long-lasting facilitation of these reflexes (LLFR) by largely unknown mechanisms. Two Sprague-Dawley substrains were used to characterize network properties involved in this serotonin (5-HT) receptor-mediated reflex plasticity. Serotonin more easily produced LLFR in one substrain and a long-lasting depression of reflexes (LLDR) in the other. Interestingly, LLFR and LLDR were bidirectionally interconvertible using 5-HT2A/2C and 5-HT1A receptor agonists, respectively, regardless of substrain. LLFR was predominantly Aβ afferent fiber mediated, consistent with prominent 5-HT2C receptor expression in the Aβ fiber projection territories (deeper spinal laminae). Reflex facilitation involved an unmasking of polysynaptic pathways and an increased receptive field size. LLFR emerged even when reflexes were evoked three to five times/h, indicating an activity independent induction. Both the NMDA and AMPA/kainate receptor-mediated components of the reflex could be facilitated, and facilitation was dependent on 5-HT receptor activation alone, not on coincident reflex activation in the presence of 5-HT. Selective blockade of GABAA and/or glycine receptors also did not prevent reflex amplification and so are not required for LLFR. Indeed, a more robust response was seen after blockade of spinal inhibition, indicating that inhibitory processes serve to limit reflex amplification. Overall we demonstrate that the serotonergic system has the capacity to induce long-lasting bidirectional changes in reflex strength in a manner that is nonassociative and independent of evoked activity or activation of ionotropic excitatory and inhibitory receptors. PMID:16033939

  17. Recombinant human betacellulin. Molecular structure, biological activities, and receptor interaction.

    PubMed

    Watanabe, T; Shintani, A; Nakata, M; Shing, Y; Folkman, J; Igarashi, K; Sasada, R

    1994-04-01

    Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR. PMID:8144591

  18. Activation and Regulation of Purinergic P2X Receptor Channels

    PubMed Central

    Coddou, Claudio; Yan, Zonghe; Obsil, Tomas; Huidobro-Toro, J. Pablo

    2011-01-01

    Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions. PMID:21737531

  19. Predicted structure of the extracellular region of ligand-gated ion-channel receptors shows SH2-like and SH3-like domains forming the ligand-binding site.

    PubMed Central

    Gready, J. E.; Ranganathan, S.; Schofield, P. R.; Matsuo, Y.; Nishikawa, K.

    1997-01-01

    Fast synaptic neurotransmission is mediated by ligand-gated ion-channel (LGIC) receptors, which include receptors for acetylcholine, serotonin, GABA, glycine, and glutamate. LGICs are pentamers with extracellular ligand-binding domains and form integral membrane ion channels that are selective for cations (acetylcholine and serotonin 5HT3 receptors) or anions (GABAA and glycine receptors and the invertebrate glutamate-binding chloride channel). They form a protein superfamily with no sequence similarity to any protein of known structure. Using a 1D-3D structure mapping approach, we have modeled the extracellular ligand-binding domain based on a significant match with the SH2 and SH3 domains of the biotin repressor structure. Refinement of the model based on knowledge of the large family of SH2 and SH3 structures, sequence alignments, and use of structure templates for loop building, allows the prediction of both monomer and pentamer models. These are consistent with medium-resolution electron microscopy structures and with experimental structure/function data from ligand-binding, antibody-binding, mutagenesis, protein-labeling and subunit-linking studies, and glycosylation sites. Also, the predicted polarity of the channel pore calculated from electrostatic potential maps of pentamer models of superfamily members is consistent with known ion selectivities. Using the glycine receptor alpha 1 subunit, which forms homopentamers, the monomeric and pentameric models define the agonist and antagonist (strychnine) binding sites to a deep crevice formed by an extended loop, which includes the invariant disulfide bridge, between the SH2 and SH3 domains. A detailed binding site for strychnine is reported that is in strong agreement with known structure/function data. A site for interaction of the extracellular ligand-binding domain with the activation of the M2 transmembrane helix is also suggested. PMID:9144769

  20. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of pr...

  1. Modulation of Toll-Like Receptor Activity by Leukocyte Ig-Like Receptors and Their Effects during Bacterial Infection

    PubMed Central

    Pilsbury, Louise E.; Allen, Rachel L.; Vordermeier, Martin

    2010-01-01

    Toll-like receptors (TLRs) are a potent trigger for inflammatory immune responses. Without tight regulation their activation could lead to pathology, so it is imperative to extend our understanding of the regulatory mechanisms that govern TLR expression and function. One family of immunoregulatory proteins which can provide a balancing effect on TLR activity are the Leukocyte Ig-like receptors (LILRs), which act as innate immune receptors for self-proteins. Here we describe the LILR family, their inhibitory effect on TLR activity in cells of the monocytic lineage, their signalling pathway, and their antimicrobial effects during bacterial infection. Agents have already been identified which enhances or inhibits LILR activity raising the future possibility that modulation of LILR function could be used as a means to modulate TLR activity. PMID:20634939

  2. Activation of endplate nicotinic acetylcholine receptors by agonists.

    PubMed

    Auerbach, Anthony

    2015-10-15

    The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly

  3. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    SciTech Connect

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.; Stomski, Frank C.; Dottore, Mara; Powell, Jason; Ramshaw, Hayley; Woodcock, Joanna M.; Xu, Yibin; Guthridge, Mark; McKinstry, William J.; Lopez, Angel F.; Parker, Michael W.

    2008-08-11

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface and functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.

  4. Shadows across mu-Star? Constitutively active mu-opioid receptors revisited.

    PubMed

    Connor, Mark

    2009-04-01

    Constitutively active mu-opioid receptors (mu* receptors) are reported to be formed following prolonged agonist treatment of cells or whole animals. mu* receptors signal in the absence of activating ligand and a blockade of mu* activation of G-proteins by naloxone and naltrexone has been suggested to underlie the profound withdrawal syndrome precipitated by these antagonists in vivo. In this issue of the Journal, Divin et al. examined whether treatment of C6 glioma cells with mu-opioid receptor agonists produced constitutively active mu-opioid receptors or other commonly reported adaptations to prolonged agonist treatment. Adenylyl cyclase superactivation was readily apparent following agonist treatment but there was no evidence of the formation of constitutively active mu-opioid receptors. This result challenges the notion that prolonged agonist exposure inevitably produces mu* receptors, and is consistent with many studies of adaptations in neurons produced by chronic agonist treatment. The investigators provide no explanation of their failure to see mu* receptors in C6 cells, but this is perhaps understandable because the molecular nature of mu* receptors remains elusive, and the precise mechanisms that lead to their formation are unknown. Without knowing exactly what mu* receptors are, how they are formed and how they signal, understanding their role in cellular adaptations to prolonged opioid treatment will remain impossible. Studies such as this should refocus attention on establishing the molecular mechanisms that underlie that phenomenon of mu* receptors. PMID:19368530

  5. Synapses, NMDA receptor activity and neuronal Aβ production in Alzheimer's disease.

    PubMed

    Bordji, Karim; Becerril-Ortega, Javier; Buisson, Alain

    2011-01-01

    A direct relationship has been established between synaptic activity and amyloid-β secretion. Dysregulation of neuronal calcium homeostasis was shown to increase production of amyloid-β, contributing to the initiation of Alzheimer's disease. Among the different routes of Ca(2+) entry, N-methyl-d-aspartate (NMDA) receptors, a subtype of ionotropic glutamate receptors, are especially involved in this process because of their ability to gate high levels of Ca(2+) influx. These receptors have been extensively studied for their crucial roles in synaptic plasticity that underlies learning and memory but also in neurotoxicity occurring during acute brain injuries and neurodegenerative diseases. For one decade, several studies provided evidence that NMDA receptor activation could have distinct consequences on neuronal fate, depending on their location. Synaptic NMDA receptor activation is neuroprotective, whereas extrasynaptic NMDA receptors trigger neuronal death and/or neurodegenerative processes. Recent data suggest that chronic activation of extrasynaptic NMDA receptors leads to a sustained neuronal amyloid-β release and could be involved in the pathogenesis of Alzheimer's disease. Thus, as for other neurological diseases, therapeutic targeting of extrasynaptic NMDA receptors could be a promising strategy. Following this concept, memantine, unlike other NMDA receptor antagonists was shown, to preferentially target the extrasynaptic NMDA receptor signaling pathways, while relatively sparing normal synaptic activity. This molecular mechanism could therefore explain why memantine is, to date, the only clinically approved NMDA receptor antagonist for the treatment of dementia. PMID:21568789

  6. Signaling Mechanism of Cannabinoid Receptor-2 Activation-Induced β-Endorphin Release.

    PubMed

    Gao, Fang; Zhang, Ling-Hong; Su, Tang-Feng; Li, Lin; Zhou, Rui; Peng, Miao; Wu, Cai-Hua; Yuan, Xiao-Cui; Sun, Ning; Meng, Xian-Fang; Tian, Bo; Shi, Jing; Pan, Hui-Lin; Li, Man

    2016-08-01

    Activation of cannabinoid receptor-2 (CB2) results in β-endorphin release from keratinocytes, which then acts on primary afferent neurons to inhibit nociception. However, the underlying mechanism is still unknown. The CB2 receptor is generally thought to couple to Gi/o to inhibit cAMP production, which cannot explain the peripheral stimulatory effects of CB2 receptor activation. In this study, we found that in a keratinocyte cell line, the Gβγ subunits from Gi/o, but not Gαs, were involved in CB2 receptor activation-induced β-endorphin release. Inhibition of MAPK kinase, but not PLC, abolished CB2 receptor activation-induced β-endorphin release. Also, CB2 receptor activation significantly increased intracellular Ca(2+). Treatment with BAPTA-AM or thapsigargin blocked CB2 receptor activation-induced β-endorphin release. Using a rat model of inflammatory pain, we showed that the MAPK kinase inhibitor PD98059 abolished the peripheral effect of the CB2 receptor agonist on nociception. We thus present a novel mechanism of CB2 receptor activation-induced β-endorphin release through Gi/o-Gβγ-MAPK-Ca(2+) signaling pathway. Our data also suggest that stimulation of MAPK contributes to the peripheral analgesic effect of CB2 receptor agonists. PMID:26108183

  7. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    SciTech Connect

    Yliniemelae, A.; Gynther, J. ); Konschin, H.; Tylli, H. ); Rouvinen, J. )

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  8. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility

    PubMed Central

    Carta, Gaspare; Artini, Paolo Giovanni

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed. PMID:27559343

  9. Peroxisome Proliferator-Activated Receptors in Female Reproduction and Fertility.

    PubMed

    Vitti, Maurizio; Di Emidio, Giovanna; Di Carlo, Michela; Carta, Gaspare; Antonosante, Andrea; Artini, Paolo Giovanni; Cimini, Annamaria; Tatone, Carla; Benedetti, Elisabetta

    2016-01-01

    Reproductive functions may be altered by the exposure to a multitude of endogenous and exogenous agents, drug or environmental pollutants, which are known to affect gene transcription through the peroxisome proliferator-activated receptors (PPARs) activation. PPARs act as ligand activated transcription factors and regulate metabolic processes such as lipid and glucose metabolism, energy homeostasis, inflammation, and cell proliferation and differentiation. All PPARs isotypes are expressed along the hypothalamic-pituitary-gonadal axis and are strictly involved in reproductive functions. Since female fertility and energy metabolism are tightly interconnected, the research on female infertility points towards the exploration of potential PPARs activating/antagonizing compounds, mainly belonging to the class of thiazolidinediones (TZDs) and fibrates, as useful agents for the maintenance of metabolic homeostasis in women with ovarian dysfunctions. In the present review, we discuss the recent evidence about PPARs expression in the hypothalamic-pituitary-gonadal axis and their involvement in female reproduction. Finally, the therapeutic potential of their manipulation through several drugs is also discussed. PMID:27559343

  10. Utilizing Chimeric Antigen Receptors to Direct Natural Killer Cell Activity

    PubMed Central

    Hermanson, David L.; Kaufman, Dan S.

    2015-01-01

    Natural killer (NK) cells represent an attractive lymphocyte population for cancer immunotherapy due to their ability to lyse tumor targets without prior sensitization and without need for human leukocyte antigens-matching. Chimeric antigen receptors (CARs) are able to enhance lymphocyte targeting and activation toward diverse malignancies. CARs consist of an external recognition domain (typically a small chain variable fragment) directed at a specific tumor antigen that is linked with one or more intracellular signaling domains that mediate lymphocyte activation. Most CAR studies have focused on their expression in T cells. However, use of CARs in NK cells is starting to gain traction because they provide a method to redirect these cells more specifically to target refractory cancers. CAR-mediated anti-tumor activity has been demonstrated using NK cell lines, as well as NK cells isolated from peripheral blood, and NK cells produced from human pluripotent stem cells. This review will outline the CAR constructs that have been reported in NK cells with a focus on comparing the use of different signaling domains in combination with other co-activating domains. PMID:25972867

  11. Visualization of Estrogen Receptor Transcriptional Activation in Zebrafish

    PubMed Central

    Halpern, Marnie E.

    2011-01-01

    Estrogens regulate a diverse range of physiological processes and affect multiple tissues. Estrogen receptors (ERs) regulate transcription by binding to DNA at conserved estrogen response elements, and such elements have been used to report ER activity in cultured cells and in transgenic mice. We generated stable, transgenic zebrafish containing five consecutive elements upstream of a c-fos minimal promoter and green fluorescent protein (GFP) to visualize and quantify transcriptional activation in live larvae. Transgenic larvae show robust, dose-dependent estrogen-dependent fluorescent labeling in the liver, consistent with er gene expression, whereas ER antagonists inhibit GFP expression. The nonestrogenic steroids dexamethasone and progesterone fail to activate GFP, confirming ER selectivity. Natural and synthetic estrogens activated the transgene with varying potency, and two chemicals, genistein and bisphenol A, preferentially induce GFP expression in the heart. In adult fish, fluorescence was observed in estrogenic tissues such as the liver, ovary, pituitary gland, and brain. Individual estrogen-responsive neurons and their projections were visualized in the adult brain, and GFP-positive neurons increased in number after 17β-estradiol exposure. The transgenic estrogen-responsive zebrafish allow ER signaling to be monitored visually and serve as in vivo sentinels for detection of estrogenic compounds. PMID:21540282

  12. Linking estrogen receptor β expression with inflammatory bowel disease activity

    PubMed Central

    Pierdominici, Marina; Maselli, Angela; Varano, Barbara; Barbati, Cristiana; Cesaro, Paola; Spada, Cristiano; Zullo, Angelo; Lorenzetti, Roberto; Rosati, Marco; Rainaldi, Gabriella; Limiti, Maria Rosaria; Guidi, Luisa

    2015-01-01

    Crohn disease (CD) and ulcerative colitis (UC) are chronic forms of inflammatory bowel disease (IBD) whose pathogenesis is only poorly understood. Estrogens have a complex role in inflammation and growing evidence suggests that these hormones may impact IBD pathogenesis. Here, we demonstrated a significant reduction (p < 0.05) of estrogen receptor (ER)β expression in peripheral blood T lymphocytes from CD/UC patients with active disease (n = 27) as compared to those in remission (n = 21) and healthy controls (n = 29). Accordingly, in a subgroup of CD/UC patients undergoing to anti-TNF-α therapy and responsive to treatment, ERβ expression was higher (p < 0.01) than that observed in not responsive patients and comparable to that of control subjects. Notably, ERβ expression was markedly decreased in colonic mucosa of CD/UC patients with active disease, reflecting the alterations observed in peripheral blood T cells. ERβ expression inversely correlated with interleukin (IL)-6 serum levels and exogenous exposure of both T lymphocytes and intestinal epithelial cells to this cytokine resulted in ERβ downregulation. These results demonstrate that the ER profile is altered in active IBD patients at both mucosal and systemic levels, at least in part due to IL-6 dysregulation, and highlight the potential exploitation of T cell-associated ERβ as a biomarker of endoscopic disease activity. PMID:26497217

  13. Pharmacologic Evaluation of Antidepressant Activity and Synthesis of 2-Morpholino-5-phenyl-6H-1,3,4-thiadiazine Hydrobromide.

    PubMed

    Sarapultsev, Alexey P; Chupakhin, Oleg N; Sarapultsev, Petr A; Sidorova, Larisa P; Tseitler, Tatiana A

    2016-01-01

    Substituted thiadiazines exert a reliable therapeutic effect in treating stress, and a schematic description of their ability to influence all aspects of a stress response has been depicted. This study was conducted to pharmacologically evaluate compound L-17, a substituted thiadiazine, (2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide) for possible anti-psychotic/antidepressant activity. Compound L-17 was synthesized by cyclocondensation of α-bromoacetophenone with the original morpholine-4-carbothionic acid hydrazide. Pharmacologic evaluations were conducted using methods described by E.F. Lavretskaya (1985), and in accordance with published guidelines for studying drugs for neuroleptic activity. Compound L-17 was evaluated for various possible mechanisms of action, including its effects on cholinergic system agonists/antagonists, dopaminergic neurotransmission, the adrenergic system, and 5-HT3 serotonin receptors. One or more of these mechanisms may be responsible for the beneficial effects shown by thiadiazine compounds in experiments conducted to evaluate their activity in models of acute stress and acute myocardial infarction. PMID:27213404

  14. Pharmacologic Evaluation of Antidepressant Activity and Synthesis of 2-Morpholino-5-phenyl-6H-1,3,4-thiadiazine Hydrobromide

    PubMed Central

    Sarapultsev, Alexey P.; Chupakhin, Oleg N.; Sarapultsev, Petr A.; Sidorova, Larisa P.; Tseitler, Tatiana A.

    2016-01-01

    Substituted thiadiazines exert a reliable therapeutic effect in treating stress, and a schematic description of their ability to influence all aspects of a stress response has been depicted. This study was conducted to pharmacologically evaluate compound L-17, a substituted thiadiazine, (2-morpholino-5-phenyl-6H-1,3,4-thiadiazine, hydrobromide) for possible anti-psychotic/antidepressant activity. Compound L-17 was synthesized by cyclocondensation of α-bromoacetophenone with the original morpholine-4-carbothionic acid hydrazide. Pharmacologic evaluations were conducted using methods described by E.F. Lavretskaya (1985), and in accordance with published guidelines for studying drugs for neuroleptic activity. Compound L-17 was evaluated for various possible mechanisms of action, including its effects on cholinergic system agonists/antagonists, dopaminergic neurotransmission, the adrenergic system, and 5-HT3 serotonin receptors. One or more of these mechanisms may be responsible for the beneficial effects shown by thiadiazine compounds in experiments conducted to evaluate their activity in models of acute stress and acute myocardial infarction. PMID:27213404

  15. Ryanodine receptor dysfunction and triggered activity in the heart.

    PubMed

    Katra, Rodolphe P; Oya, Toshiyuki; Hoeker, Gregory S; Laurita, Kenneth R

    2007-05-01

    Arrhythmogenesis has been increasingly linked to cardiac ryanodine receptor (RyR) dysfunction. However, the mechanistic relationship between abnormal RyR function and arrhythmogenesis in the heart is not clear. We hypothesize that, under abnormal RyR conditions, triggered activity will be caused by spontaneous calcium release (SCR) events that depend on transmural heterogeneities of calcium handling. We performed high-resolution optical mapping of intracellular calcium and transmembrane potential in the canine left ventricular wedge preparation (n = 28). Rapid pacing was used to initiate triggered activity under normal and abnormal RyR conditions induced by FKBP12.6 dissociation and beta-adrenergic stimulation (20-150 microM rapamycin, 0.2 microM isoproterenol). Under abnormal RyR conditions, almost all preparations experienced SCRs and triggered activity, in contrast to control, rapamycin, or isoproterenol conditions alone. Furthermore, under abnormal RyR conditions, complex arrhythmias (monomorphic and polymorphic tachycardia) were commonly observed. After washout of rapamycin and isoproterenol, no triggered activity was observed. Surprisingly, triggered activity and SCRs occurred preferentially near the epicardium but not the endocardium (P < 0.01). Interestingly, the occurrence of triggered activity and SCR events could not be explained by cytoplasmic calcium levels, but rather by fast calcium reuptake kinetics. These data suggest that, under abnormal RyR conditions, triggered activity is caused by multiple SCR events that depend on the faster calcium reuptake kinetics near the epicardium. Furthermore, multiple regions of SCR may be a mechanism for multifocal arrhythmias associated with RyR dysfunction. PMID:17189349

  16. His499 Regulates Dimerization and Prevents Oncogenic Activation by Asparagine Mutations of the Human Thrombopoietin Receptor.

    PubMed

    Leroy, Emilie; Defour, Jean-Philippe; Sato, Takeshi; Dass, Sharmila; Gryshkova, Vitalina; Shwe, Myat M; Staerk, Judith; Constantinescu, Stefan N; Smith, Steven O

    2016-02-01

    Ligand binding to the extracellular domain of the thrombopoietin receptor (TpoR) imparts a specific orientation on the transmembrane (TM) and intracellular domains of the receptors that is required for physiologic activation via receptor dimerization. To map the inactive and active dimeric orientations of the TM helices, we performed asparagine (Asn)-scanning mutagenesis of the TM domains of the murine and human TpoR. Substitution of Asn at only one position (S505N) activated the human receptor, whereas Asn substitutions at several positions activated the murine receptor. Second site mutational studies indicate that His(499) near the N terminus of the TM domain is responsible for protecting the human receptor from activation by Asn mutations. Structural studies reveal that the sequence preceding His(499) is helical in the murine receptor but non-helical in peptides corresponding to the TM domain of the inactive human receptor. The activating S505N mutation and the small molecule agonist eltrombopag both induce helix in this region of the TM domain and are associated with dimerization and activation of the human receptor. Thus, His(499) regulates the activation of human TpoR and provides additional protection against activating mutations, such as oncogenic Asn mutations in the TM domain. PMID:26627830

  17. Chronic activation of CB2 cannabinoid receptors in the hippocampus increases excitatory synaptic transmission

    PubMed Central

    Kim, Jimok; Li, Yong

    2015-01-01

    The roles of CB1 cannabinoid receptors in regulating neuronal activity have been extensively characterized. Although early studies show that CB1 receptors are present in the nervous system and CB2 cannabinoid receptors are in the immune system, recent evidence indicates that CB2 receptors are also expressed in the brain. Activation or blockade of CB2 receptors in vivo induces neuropsychiatric effects, but the cellular mechanisms of CB2 receptor function are unclear. The aim of this study is to determine how activation of CB2 receptors present in the hippocampus regulates synaptic function. Here, we show that when organotypic cultures of rodent hippocampal slices were treated with a CB2 receptor agonist (JWH133 or GP1a) for 7–10 days, quantal glutamate release became more frequent and spine density was increased via extracellular signal-regulated kinases. Chronic intraperitoneal injection of JWH133 into mice also increased excitatory synaptic transmission. These effects were blocked by a CB2 receptor antagonist (SR144528) or absent from hippocampal slices of CB2 receptor knock-out mice. This study reveals a novel cellular function of CB2 cannabinoid receptors in the hippocampus and provides insights into how cannabinoid receptor subtypes diversify the roles of cannabinoids in the brain. PMID:25504573

  18. Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation.

    PubMed

    Golder, Francis J; Ranganathan, Lavanya; Satriotomo, Irawan; Hoffman, Michael; Lovett-Barr, Mary Rachael; Watters, Jyoti J; Baker-Herman, Tracy L; Mitchell, Gordon S

    2008-02-27

    Acute intermittent hypoxia elicits a form of spinal, brain-derived neurotrophic factor (BDNF)-dependent respiratory plasticity known as phrenic long-term facilitation. Ligands that activate G(s)-protein-coupled receptors, such as the adenosine 2a receptor, mimic the effects of neurotrophins in vitro by transactivating their high-affinity receptor tyrosine kinases, the Trk receptors. Thus, we hypothesized that A2a receptor agonists would elicit phrenic long-term facilitation by mimicking the effects of BDNF on TrkB receptors. Here we demonstrate that spinal A2a receptor agonists transactivate TrkB receptors in the rat cervical spinal cord near phrenic motoneurons, thus inducing long-lasting (hours) phrenic motor facilitation. A2a receptor activation increased phosphorylation and new synthesis of an immature TrkB protein, induced TrkB signaling through Akt, and strengthened synaptic pathways to phrenic motoneurons. RNA interference targeting TrkB mRNA demonstrated that new TrkB protein synthesis is necessary for A2a-induced phrenic motor facilitation. A2a receptor activation also increased breathing in unanesthetized rats, and improved breathing in rats with cervical spinal injuries. Thus, small, highly permeable drugs (such as adenosine receptor agonists) that transactivate TrkB receptors may provide an effective therapeutic strategy in the treatment of patients with ventilatory control disorders, such as obstructive sleep apnea, or respiratory insufficiency after spinal injury or during neurodegenerative diseases. PMID:18305238

  19. Activity of new NOP receptor ligands in a rat peripheral mononeuropathy model: Potentiation of Morphine anti-allodynic activity by NOP receptor antagonists

    PubMed Central

    Khroyan, Taline V.; Polgar, Willma E.; Orduna, Juan; Jiang, Faming; Olsen, Cris; Toll, Lawrence; Zaveri, Nurulain T.

    2009-01-01

    The effect of new NOP receptor agonists and antagonists in the rat chronic constriction injury model was investigated. Intraperitoneally administered NOP receptor agonist SR14150 and antagonists SR16430 and SR14148, had no effect on mechanical allodynia when given alone. The nonselective NOP/mu-opioid receptor agonist SR16435, however, produced an anti-allodynic response, similar to morphine and reversible by naloxone. Notably, co-administration of the NOP receptor antagonists potentiated the anti-allodynic activity of both morphine and SR16435. Increased levels of the NOP receptor are implicated in the reduced efficacy of morphine in neuropathic pain. Our results suggest the utility of NOP receptor antagonists for potentiating opioid efficacy in chronic pain. PMID:19285491

  20. Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity.

    PubMed

    Krabbe, Grietje; Matyash, Vitali; Pannasch, Ulrike; Mamer, Lauren; Boddeke, Hendrikus W G M; Kettenmann, Helmut

    2012-03-01

    Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP-/+ mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties. PMID:22198120

  1. Angiotensin II AT1 receptor constitutive activation: from molecular mechanisms to pathophysiology.

    PubMed

    Petrel, Christophe; Clauser, Eric

    2009-04-29

    Mutations activating the angiotensin II AT(1) receptor are important to identify and characterize because they give access to the activation mechanisms of this G protein coupled receptor and help to characterize the signaling pathways and the potential pathophysiology of this receptor. The different constitutively activated mutations of the AT(1) receptor are mostly localized in transmembrane domains (TM) and their characterization demonstrated that release of intramolecular constraints and movements among these TM are a necessary step for receptor activation. These mutations constitutively activate Gq linked signaling pathways, receptor internalization and maybe the G protein-independent signaling pathways. Expression of such mutations in mice is linked to hypertension and cardiovascular diseases, but such natural mutations have not been identified in human pathology. PMID:19061936

  2. Death-receptor activation halts clathrin-dependent endocytosis

    PubMed Central

    Austin, Cary D.; Lawrence, David A.; Peden, Andrew A.; Varfolomeev, Eugene E.; Totpal, Klara; De Mazière, Ann M.; Klumperman, Judith; Arnott, David; Pham, Victoria; Scheller, Richard H.; Ashkenazi, Avi

    2006-01-01

    Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2α between functionally distinct domains, whereas effector caspases processed clathrin’s heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death. PMID:16801533

  3. Determination of the architecture of ionotropic receptors using AFM imaging.

    PubMed

    Barrera, Nelson P; Henderson, Robert M; Edwardson, J Michael

    2008-04-01

    Fast neurotransmission in the nervous system is mediated by ionotropic receptors, all of which contain several subunits surrounding an integral ion channel. There are three major families of ionotropic receptors: the 'Cys-loop' receptors (including the nicotinic receptor for acetylcholine, the 5-HT(3) receptor, the GABA(A) receptor and the glycine receptor), the glutamate receptors (including the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, kainate and N-methyl-D: -aspartic acid receptors) and the P2X receptors for adenosine triphosphate. These receptors are often built from multiple types of subunit, raising the question of the stoichiometry and subunit arrangement within the receptors. This question is of therapeutic significance because in some cases drug-binding sites are located at subunit-subunit interfaces. In this paper, we describe a general method, based on atomic force microscopy imaging, to solve the architecture of multi-subunit proteins, such as the ionotropic receptors. Specific epitope tags are engineered onto each receptor subunit. The subunits are then expressed exogenously in cultured cells, and the receptors are isolated from detergent extracts of membrane fractions by affinity chromatography. The receptors are imaged both alone and in complex with anti-epitope antibodies. The size of the imaged particles provides an estimate of the subunit stoichiometry, whereas the geometry of the receptor-antibody complexes produces more detailed information about the receptor architecture. We use an automated, unbiased system to identify receptors and receptor-antibody complexes and to determine the geometry of the complexes. We are also able to determine the orientation of the receptors on the mica substrate, which will allow us to solve the subunit arrangement within receptors, such as the GABA(A) receptor, which contain three types of subunits. PMID:18026748

  4. Characterization of the intrinsic activity for a novel class of cannabinoid receptor ligands: Indole Quinuclidine analogues

    PubMed Central

    Franks, Lirit N.; Ford, Benjamin M.; Madadi, Nikhil R.; Penthala, Narsimha R.; Crooks, Peter A.; Prather, Paul L.

    2014-01-01

    Our laboratory recently reported that a group of novel indole quinuclidine analogues bind with nanomolar affinity to cannabinoid type-1 and type-2 receptors. This study characterized the intrinsic activity of these compounds by determining whether they exhibit agonist, antagonist, or inverse agonist activity at cannabinoid type-1 and/or type-2 receptors. Cannabinoid receptors activate Gi/Go-proteins that then proceed to inhibit activity of the downstream intracellular effector adenylyl cyclase. Therefore, intrinsic activity was quantified by measuring the ability of compounds to modulate levels of intracellular cAMP in intact cells. Concerning cannabinoid type-1 receptors endogenously expressed in Neuro2A cells, a single analogue exhibited agonist activity, while eight acted as neutral antagonists and two possessed inverse agonist activity. For cannabinoid type-2 receptors stably expressed in CHO cells, all but two analogues acted as agonists; these two exceptions exhibited inverse agonist activity. Confirming specificity at cannabinoid type-1 receptors, modulation of adenylyl cyclase activity by all proposed agonists and inverse agonists was blocked by co-incubation with the neutral cannabinoid type-1 antagonist O-2050. All proposed cannabinoid type-1 receptor antagonists attenuated adenylyl cyclase modulation by cannabinoid agonist CP-55,940. Specificity at cannabinoid type-2 receptors was confirmed by failure of all compounds to modulate adenylyl cyclase activity in CHO cells devoid of cannabinoid type-2 receptors. Further characterization of select analogues demonstrated concentration-dependent modulation of adenylyl cyclase activity with potencies similar to their respective affinities for cannabinoid receptors. Therefore, indole quinuclidines are a novel structural class of compounds exhibiting high affinity and a range of intrinsic activity at cannabinoid type-1 and type-2 receptors. PMID:24858620

  5. Odorant receptor-mediated sperm activation in disease vector mosquitoes

    PubMed Central

    Pitts, R. Jason; Liu, Chao; Zhou, Xiaofan; Malpartida, Juan C.; Zwiebel, Laurence J.

    2014-01-01

    Insects, such as the malaria vector mosquito, Anopheles gambiae, depend upon chemoreceptors to respond to volatiles emitted from a range of environmental sources, most notably blood meal hosts and oviposition sites. A subset of peripheral signaling pathways involved in these insect chemosensory-dependent behaviors requires the activity of heteromeric odorant receptor (OR) ion channel complexes and ligands for numerous A. gambiae ORs (AgOrs) have been identified. Although AgOrs are expressed in nonhead appendages, studies characterizing potential AgOr function in nonolfactory tissues have not been conducted. In the present study, we explore the possibility that AgOrs mediate responses of spermatozoa to endogenous signaling molecules in A. gambiae. In addition to finding AgOr transcript expression in testes, we show that the OR coreceptor, AgOrco, is localized to the flagella of A. gambiae spermatozoa where Orco-specific agonists, antagonists, and other odorant ligands robustly activate flagella beating in an Orco-dependent process. We also demonstrate Orco expression and Orco-mediated activation of spermatozoa in the yellow fever mosquito, Aedes aegypti. Moreover, we find Orco localization in testes across distinct insect taxa and posit that OR-mediated responses in spermatozoa may represent a general characteristic of insect reproduction and an example of convergent evolution. PMID:24550284

  6. A mechanism of intracellular P2X receptor activation.

    PubMed

    Sivaramakrishnan, Venketesh; Fountain, Samuel J

    2012-08-17

    P2X receptors (P2XRs) are ATP-activated calcium-permeable ligand-gated ion channels traditionally viewed as sensors of extracellular ATP during diverse physiological processes including pain, inflammation, and taste. However, in addition to a cell surface residency P2XRs also populate the membranes of intracellular compartments, including mammalian lysosomes, phagosomes, and the contractile vacuole (CV) of the amoeba Dictyostelium. The function of intracellular P2XRs is unclear and represents a major gap in our understanding of ATP signaling. Here, we exploit the genetic versatility of Dictyostelium to investigate the effects of physiological concentrations of ATP on calcium signaling in isolated CVs. Within the CV, an acidic calcium store, P2XRs are orientated to sense luminal ATP. Application of ATP to isolated vacuoles leads to luminal translocation of ATP and release of calcium. Mechanisms of luminal ATP translocation and ATP-evoked calcium release share common pharmacology, suggesting that they are linked processes. The ability of ATP to mobilize stored calcium is reduced in vacuoles isolated from P2X(A)R knock-out amoeba and ablated in cells devoid of P2XRs. Pharmacological inhibition of luminal ATP translocation or depletion of CV calcium attenuates CV function in vivo, manifesting as a loss of regulatory cell volume decrease following osmotic swelling. We propose that intracellular P2XRs regulate vacuole activity by acting as calcium release channels, activated by translocation of ATP into the vacuole lumen. PMID:22736763

  7. SENESCENCE-ASSOCIATED DECLINE IN HEPATIC PEROXISOMAL ENZYME ACTIVITIES CORRESPONDS WITH DIMINISHED LEVELS OF RETINOID X RECEPTOR ALPHA, BUT NOT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR ALPHA1

    EPA Science Inventory

    Abstract

    Aging is associated with alterations in hepatic peroxisomal metabolism and susceptibility to hepatocarcinogenecity produced by agonists of peroxisome proliferator-activated receptor alpha (PPARa). Mechanisms involved in these effects are not well understood. Howev...

  8. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    PubMed

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms. PMID:25330347

  9. Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

    PubMed

    Neale, Chris; Herce, Henry D; Pomès, Régis; García, Angel E

    2015-10-20

    G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated β2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization. PMID:26488656

  10. Activation of 5-hyrdoxytryptamine 7 receptors within the rat nucleus tractus solitarii modulates synaptic properties.

    PubMed

    Matott, Michael P; Kline, David D

    2016-03-15

    Serotonin (5-HT) is a potent neuromodulator with multiple receptor types within the cardiorespiratory system, including the nucleus tractus solitarii (nTS) - the central termination site of visceral afferent fibers. The 5-HT7 receptor facilitates cardiorespiratory reflexes through its action in the brainstem and likely in the nTS. However, the mechanism and site of action for these effects is not clear. In this study, we examined the expression and function of 5-HT7 receptors in the nTS of Sprague-Dawley rats. 5-HT7 receptor mRNA and protein were identified across the rostrocaudal extent of the nTS. To determine 5-HT7 receptor function, we examined nTS synaptic properties following 5-HT7 receptor activation in monosynaptic nTS neurons in the in vitro brainstem slice preparation. Application of 5-HT7 receptor agonists altered tractus solitarii evoked and spontaneous excitatory postsynaptic currents which were attenuated with a selective 5-HT7 receptor antagonist. 5-HT7 receptor-mediated changes in excitatory postsynaptic currents were also altered by block of 5-HT1A and GABAA receptors. Interestingly, 5-HT7 receptor activation also reduced the amplitude but not frequency of GABAA-mediated inhibitory currents. Together these results indicate a complex role for 5-HT7 receptors in the nTS that mediate its diverse effects on cardiorespiratory parameters. PMID:26779891

  11. Triclocarban Mediates Induction of Xenobiotic Metabolism through Activation of the Constitutive Androstane Receptor and the Estrogen Receptor Alpha

    PubMed Central

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M.; Hammock, Bruce; Tukey, Robert H.

    2012-01-01

    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

  12. Activation of retinoid X receptors induces apoptosis in HL-60 cell lines.

    PubMed Central

    Nagy, L; Thomázy, V A; Shipley, G L; Fésüs, L; Lamph, W; Heyman, R A; Chandraratna, R A; Davies, P J

    1995-01-01

    Retinoids induce myeloblastic leukemia (HL-60) cells to differentiate into granulocytes, which subsequently die by apoptosis. Retinoid action is mediated through at least two classes of nuclear receptors: retinoic acid receptors, which bind both all-trans retinoic acid and 9-cis retinoic acid, and retinoid X receptors, which bind only 9-cis retinoic acid. Using receptor-selective synthetic retinoids and HL-60 cell sublines with different retinoid responsiveness, we have investigated the contribution that each class of receptors makes to the processes of cellular differentiation and death. Our results demonstrate that ligand activation of retinoic acid receptors is sufficient to induce differentiation, whereas ligand activation of retinoid X receptors is essential for the induction of apoptosis in HL-60 cell lines. PMID:7791761

  13. Investigating real-time activation of adenosine receptors by bioluminescence resonance energy transfer technique

    NASA Astrophysics Data System (ADS)

    Huang, Yimei; Yang, Hongqin; Zheng, Liqin; Chen, Jiangxu; Wang, Yuhua; Li, Hui; Xie, Shusen

    2013-02-01

    Adenosine receptors play important roles in many physiological and pathological processes, for example regulating myocardial oxygen consumption and the release of neurotransmitters. The activations of adenosine receptors have been studied by some kinds of techniques, such as western blot, immunohistochemistry, etc. However, these techniques cannot reveal the dynamical response of adenosine receptors under stimulation. In this paper, bioluminescence resonance energy transfer technique was introduced to study the real-time activation of adenosine receptors by monitoring the dynamics of cyclic adenosine monophosphate (cAMP) level. The results showed that there were significant differences between adenosine receptors on real-time responses under stimulation. Moreover, the dynamics of cAMP level demonstrated that competition between adenosine receptors existed. Taken together, our study indicates that monitoring the dynamics of cAMP level using bioluminescence resonance energy transfer technique could be one potential approach to investigate the mechanism of competitions between adenosine receptors.

  14. The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

    PubMed Central

    Woolley, Michael. J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.

    2013-01-01

    The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. PMID:24047872

  15. Chronic activation of 5-HT4 receptors or blockade of 5-HT6 receptors improve memory performances.

    PubMed

    Quiedeville, Anne; Boulouard, Michel; Hamidouche, Katia; Da Silva Costa-Aze, Virginie; Nee, Gerald; Rochais, Christophe; Dallemagne, Patrick; Fabis, Frédéric; Freret, Thomas; Bouet, Valentine

    2015-10-15

    5-HT4 and 5-HT6 serotonergic receptors are located in brain structures involved in memory processes. Neurochemical and behavioural studies have demonstrated that acute activation of 5-HT4 receptors (5-HT4R) or blockade of 5-HT6 receptors (5-HT6R) improves memory. To evaluate the potential of these two receptors as targets in the treatment of memory disorders encountered in several situations (ageing, Alzheimer's disease, schizophrenia, etc.), it is necessary to assess whether their beneficial effects occur after chronic administration, and if such treatment induces adverse effects. The goal of this study was to assess the effects of chronic 5-HT4R or 5-HT6R modulation on recognition memory, and to observe the possible manifestation of side effects (modification of weight gain, locomotor activity or exploratory behaviour, etc.). Mice were treated for 14 days with a 5-HT4R partial agonist (RS-67333) or a 5-HT6R antagonist (SB-271046) at increasing doses. Memory performances, locomotor activity, and exploration were assessed. Both chronic 5-HT4R activation and 5-HT6R blockade extended memory traces in an object recognition test, and were not associated with any adverse effects in the parameters assessed. Chronic modulation of one or both of these receptors thus seems promising as a potential strategy for the treatment memory deficits. PMID:26187692

  16. Role of receptors in Bacillus thuringiensis crystal toxin activity.

    PubMed

    Pigott, Craig R; Ellar, David J

    2007-06-01

    Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death. PMID:17554045

  17. The influence of selective vitamin D receptor activator paricalcitol on cardiovascular system and cardiorenal protection

    PubMed Central

    Duplancic, Darko; Cesarik, Marijan; Poljak, Nikola Kolja; Radman, Maja; Kovacic, Vedran; Radic, Josipa; Rogosic, Veljko

    2013-01-01

    The ubiquitous distribution of vitamin D receptors in the human body is responsible for the pleiotropic effects of vitamin D-receptor activation. We discuss the possible beneficial effects of a selective activator of vitamin D receptor, paricalcitol, on the cardiovascular system in chronic heart failure patients and chronic kidney patients, in light of new trials. Paricalcitol should provide additional clinical benefits over the standard treatment for chronic kidney and heart failure, especially in cases of cardiorenal syndrome. PMID:23430986

  18. Analysis of the Heat Shock Response in Mouse Liver Reveals Transcriptional Dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARα)

    EPA Science Inventory

    BACKGROUND: The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARalpha) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by h...

  19. Adipocyte insulin receptor activity maintains adipose tissue mass and lifespan.

    PubMed

    Friesen, Max; Hudak, Carolyn S; Warren, Curtis R; Xia, Fang; Cowan, Chad A

    2016-08-01

    Type 2 diabetes follows a well-defined progressive pathogenesis, beginning with insulin resistance in metabolic tissues such as the adipose. Intracellular signaling downstream of insulin receptor activation regulates critical metabolic functions of adipose tissue, including glucose uptake, lipogenesis, lipolysis and adipokine secretion. Previous studies have used the aP2 promoter to drive Cre recombinase expression in adipose tissue. Insulin receptor (IR) knockout mice created using this aP2-Cre strategy (FIRKO mice) were protected from obesity and glucose intolerance. Later studies demonstrated the promiscuity of the aP2 promoter, casting doubts upon the tissue specificity of aP2-Cre models. It is our goal to use the increased precision of the Adipoq promoter to investigate adipocyte-specific IR function. Towards this end we generated an adipocyte-specific IR knockout (AIRKO) mouse using an Adipoq-driven Cre recombinase. Here we report AIRKO mice are less insulin sensitive throughout life, and less glucose tolerant than wild-type (WT) littermates at the age of 16 weeks. In contrast to WT littermates, the insulin sensitivity of AIRKO mice is unaffected by age or dietary regimen. At any age, AIRKO mice are comparably insulin resistant to old or obese WT mice and have a significantly reduced lifespan. Similar results were obtained when these phenotypes were re-examined in FIRKO mice. We also found that the AIRKO mouse is protected from high-fat diet-induced weight gain, corresponding with a 90% reduction in tissue weight of major adipose depots compared to WT littermates. Adipose tissue mass reduction is accompanied by hepatomegaly and increased hepatic steatosis. These data indicate that adipocyte IR function is crucial to systemic energy metabolism and has profound effects on adiposity, hepatic homeostasis and lifespan. PMID:27246738

  20. Cannabinoid Receptor Activation Shifts Temporally Engendered Patterns of Dopamine Release

    PubMed Central

    Oleson, Erik B; Cachope, Roger; Fitoussi, Aurelie; Tsutsui, Kimberly; Wu, Sharon; Gallegos, Jacqueline A; Cheer, Joseph F

    2014-01-01

    The ability to discern temporally pertinent environmental events is essential for the generation of adaptive behavior in conventional tasks, and our overall survival. Cannabinoids are thought to disrupt temporally controlled behaviors by interfering with dedicated brain timing networks. Cannabinoids also increase dopamine release within the mesolimbic system, a neural pathway generally implicated in timing behavior. Timing can be assessed using fixed-interval (FI) schedules, which reinforce behavior on the basis of time. To date, it remains unknown how cannabinoids modulate dopamine release when responding under FI conditions, and for that matter, how subsecond dopamine release is related to time in these tasks. In the present study, we hypothesized that cannabinoids would accelerate timing behavior in an FI task while concurrently augmenting a temporally relevant pattern of dopamine release. To assess this possibility, we measured subsecond dopamine concentrations in the nucleus accumbens while mice responded for food under the influence of the cannabinoid agonist WIN 55 212-2 in an FI task. Our data reveal that accumbal dopamine concentrations decrease proportionally to interval duration—suggesting that dopamine encodes time in FI tasks. We further demonstrate that WIN 55 212-2 dose-dependently increases dopamine release and accelerates a temporal behavioral response pattern in a CB1 receptor-dependent manner—suggesting that cannabinoid receptor activation modifies timing behavior, in part, by augmenting time-engendered patterns of dopamine release. Additional investigation uncovered a specific role for endogenous cannabinoid tone in timing behavior, as elevations in 2-arachidonoylglycerol, but not anandamide, significantly accelerated the temporal response pattern in a manner akin to WIN 55 212-2. PMID:24345819

  1. Kallikrein Promotes Inflammation in Human Dental Pulp Cells Via Protease-Activated Receptor-1.

    PubMed

    Hayama, Tomomi; Kamio, Naoto; Okabe, Tatsu; Muromachi, Koichiro; Matsushima, Kiyoshi

    2016-07-01

    Plasma kallikrein (KLKB1), a serine protease, cleaves high-molecular weight kininogen to produce bradykinin, a potent vasodilator and pro-inflammatory peptide. In addition, KLKB1 activates plasminogen and other leukocyte and blood coagulation factors and processes pro-enkephalin, prorenin, and C3. KLKB1 has also been shown to cleave protease-activated receptors in vascular smooth muscle cells to regulate the expression of epidermal growth factor receptor. In this study, we investigated KLKB1-dependent inflammation and activation of protease-activated receptor-1 in human dental pulp cells. These cells responded to KLKB1 stimulation by increasing intracellular Ca(2+) , upregulating cyclooxygenase-2, and secreting prostaglandin E2 . Remarkably, SCH79797, an antagonist of protease-activated receptor-1, blocked these effects. Thus, these data indicate that KLKB1 induces inflammatory reactions in human dental tissues via protease-activated receptor 1. J. Cell. Biochem. 117: 1522-1528, 2016. © 2015 Wiley Periodicals, Inc. PMID:26566265

  2. DIFFERENTIAL TOLL-LIKE RECEPTOR ACTIVATION IN LUNG ISCHEMIA REPERFUSION INJURY

    PubMed Central

    Phelan, Patrick; Merry, Heather E.; Hwang, Billanna; Mulligan, Michael S.

    2015-01-01

    Objective The requirement for toll-like receptors in lung ischemia reperfusion injury (LIRI) has been demonstrated but not fully characterized. We have previously reported that toll-like receptor-4 is required by alveolar macrophages but not pulmonary endothelial or epithelial cells for the development of LIRI. Additionally, we have demonstrated differential patterns of mitogen-activated protein kinase activation and cytokine release in these cell types during LIRI. We sought to determine whether the differences in their activation responses related to cell specific toll-like receptor activation requirements. Methods Primary cultures of alveolar macrophages, pulmonary endothelial, and immortalized epithelial cells were pretreated with toll-like receptor-2 or -4 short interference (si)RNA prior to hypoxia and reoxygenation. Cell lysates and media were analyzed for receptor knockdown, mitogen-activated protein kinase activation, and cytokine production. Rats were pretreated with toll-like receptor-2 or -4 siRNA prior to lung ischemia reperfusion and changes in lung vascular permeability were assessed. Results Toll-like receptor-2 knockdown in alveolar macrophages did not affect mitogen-activated protein kinase phosphorylation or cytokine secretion. Conversely, toll-like receptor-2 knockdown in pulmonary endothelial and epithelial cells demonstrated significant reductions in ERK 1/2 activation and cytokine secretion. Toll-like receptor-4, but not toll-like receptor-2, decreased lung permeability index in LIRI. Conclusions Differential toll-like receptor signaling and mitogen-activated protein kinase activation in response to LIRI appear to be cell specific. siRNA provides an outstanding tool for examination of the underlying mechanism. PMID:25911179

  3. Peroxisome proliferator-activated receptor ɣ activation induces 11β-hydroxysteroid dehydrogenase type 1 activity in human alternative macrophages

    PubMed Central

    Chinetti-Gbaguidi, Giulia; Bouhlel, Mohamed Amine; Copin, Corinne; Duhem, Christian; Derudas, Bruno; Neve, Bernardette; Noel, Benoit; Eeckhoute, Jerome; Lefebvre, Philippe; Seckl, Jonathan R.; Staels, Bart

    2012-01-01

    Objectives 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) catalyses the intracellular reduction of inactive cortisone to active cortisol, the natural ligand activating the glucocorticoid receptor (GR). Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a nuclear receptor controlling inflammation, lipid metabolism and the macrophage polarization state. In this study, we investigated the impact of macrophage polarization on the expression and activity of 11β-HSD1 and the role of PPAR therein. Methods and Results 11β-HSD1 gene expression is higher in pro-inflammatory M1 and anti-inflammatory M2 macrophages than in resting macrophages (RM), whereas its activity is highest in M2 macrophages. Interestingly, PPARγ activation induces 11β-HSD1 enzyme activity in M2 macrophages, but not in RM or M1 macrophages. Consequently, human M2 macrophages displayed enhanced responsiveness to the 11β-HSD1 substrate cortisone, an effect amplified by PPAR -induction of 11β-HSD1 activity, as illustrated by an increased expression of GR target genes. Conclusions Our data identify a positive cross-talk between PPARγ and GR in human M2 macrophages via the induction of 11β-HSD1 expression and activity. PMID:22207732

  4. Molecular vibration-activity relationship in the agonism of adenosine receptors.

    PubMed

    Chee, Hyun Keun; Oh, S June

    2013-12-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  5. Molecular Vibration-Activity Relationship in the Agonism of Adenosine Receptors

    PubMed Central

    Chee, Hyun Keun

    2013-01-01

    The molecular vibration-activity relationship in the receptor-ligand interaction of adenosine receptors was investigated by structure similarity, molecular vibration, and hierarchical clustering in a dataset of 46 ligands of adenosine receptors. The resulting dendrogram was compared with those of another kind of fingerprint or descriptor. The dendrogram result produced by corralled intensity of molecular vibrational frequency outperformed four other analyses in the current study of adenosine receptor agonism and antagonism. The tree that was produced by clustering analysis of molecular vibration patterns showed its potential for the functional classification of adenosine receptor ligands. PMID:24465242

  6. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease

    PubMed Central

    Azhar, Salman

    2011-01-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/β and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol metabolism, where imbalance can lead to obesity, T2DM and CVD. They are also drug targets, and currently, PPARα (fibrates) and PPARγ (thiazolodinediones) agonists are in clinical use for treating dyslipidemia and T2DM, respectively. These metabolic characteristics of the PPARs, coupled with their involvement in metabolic diseases, mean extensive efforts are underway worldwide to develop new and efficacious PPAR-based therapies for the treatment of additional maladies associated with the MetS. This article presents an overview of the functional characteristics of three PPAR isotypes, discusses recent advances in our understanding of the diverse biological actions of PPARs, particularly in the vascular system, and summarizes the developmental status of new single, dual, pan (multiple) and partial PPAR agonists for the clinical management of key components of MetS, T2DM and CVD. It also summarizes the clinical outcomes from various clinical trials aimed at evaluating the atheroprotective actions of currently used fibrates and thiazolodinediones. PMID:20932114

  7. Peroxisome proliferator-activated receptors, metabolic syndrome and cardiovascular disease.

    PubMed

    Azhar, Salman

    2010-09-01

    Metabolic syndrome (MetS) is a constellation of risk factors including insulin resistance, central obesity, dyslipidemia and hypertension that markedly increase the risk of Type 2 diabetes (T2DM) and cardiovascular disease (CVD). The peroxisome proliferators-activated receptor (PPAR) isotypes, PPARα, PPARδ/ß and PPARγ are ligand-activated nuclear transcription factors, which modulate the expression of an array of genes that play a central role in regulating glucose, lipid and cholesterol metabolism, where imbalance can lead to obesity, T2DM and CVD. They are also drug targets, and currently, PPARα (fibrates) and PPARγ (thiazolodinediones) agonists are in clinical use for treating dyslipidemia and T2DM, respectively. These metabolic characteristics of the PPARs, coupled with their involvement in metabolic diseases, mean extensive efforts are underway worldwide to develop new and efficacious PPAR-based therapies for the treatment of additional maladies associated with the MetS. This article presents an overview of the functional characteristics of three PPAR isotypes, discusses recent advances in our understanding of the diverse biological actions of PPARs, particularly in the vascular system, and summarizes the developmental status of new single, dual, pan (multiple) and partial PPAR agonists for the clinical management of key components of MetS, T2DM and CVD. It also summarizes the clinical outcomes from various clinical trials aimed at evaluating the atheroprotective actions of currently used fibrates and thiazolodinediones. PMID:20932114

  8. The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

    PubMed Central

    Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

    2015-01-01

    ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

  9. Endothelial Cells Promote Pigmentation through Endothelin Receptor B Activation.

    PubMed

    Regazzetti, Claire; De Donatis, Gian Marco; Ghorbel, Houda Hammami; Cardot-Leccia, Nathalie; Ambrosetti, Damien; Bahadoran, Philippe; Chignon-Sicard, Bérengère; Lacour, Jean-Philippe; Ballotti, Robert; Mahns, Andre; Passeron, Thierry

    2015-12-01

    Findings of increased vascularization in melasma lesions and hyperpigmentation in acquired bilateral telangiectatic macules suggested a link between pigmentation and vascularization. Using high-magnification digital epiluminescence dermatoscopy, laser confocal microscopy, and histological examination, we showed that benign vascular lesions of the skin have restricted but significant hyperpigmentation compared with the surrounding skin. We then studied the role of microvascular endothelial cells in regulating skin pigmentation using an in vitro co-culture model using endothelial cells and melanocytes. These experiments showed that endothelin 1 released by microvascular endothelial cells induces increased melanogenesis signaling, characterized by microphthalmia-associated transcription factor phosphorylation, and increased tyrosinase and dopachrome tautomerase levels. Immunostaining for endothelin 1 in vascular lesions confirmed the increased expression on the basal layer of the epidermis above small vessels compared with perilesional skin. Endothelin acts through the activation of endothelin receptor B and the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK)1/2, and p38, to induce melanogenesis. Finally, culturing of reconstructed skin with microvascular endothelial cells led to increased skin pigmentation that could be prevented by inhibiting EDNRB. Taken together these results demonstrated the role of underlying microvascularization in skin pigmentation, a finding that could open new fields of research for regulating physiological pigmentation and for treating pigmentation disorders such as melasma. PMID:26308584

  10. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

  11. Activated Protein C Enhances Human Keratinocyte Barrier Integrity via Sequential Activation of Epidermal Growth Factor Receptor and Tie2*

    PubMed Central

    Xue, Meilang; Chow, Shu-Oi; Dervish, Suat; Chan, Yee-Ka Agnes; Julovi, Sohel M.; Jackson, Christopher J.

    2011-01-01

    Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability. PMID:21173154

  12. Evidence that mCPP may have behavioural effects mediated by central 5-HT1C receptors.

    PubMed Central

    Kennett, G. A.; Curzon, G.

    1988-01-01

    1. The effects of 1-(3-chlorophenyl)piperazine (mCPP) and 1-[3-(trifluoromethyl)phenyl] piperazine (TFMPP) on activity of rats in a novel cage, and on the rotorod and elevated bar co-ordination tests was examined. 2. Peripherally administered mCPP and TFMPP dose-dependently reduced locomotion, rearing, and feeding scores but not grooming of freely fed rats placed in a novel observation cage. Yawning behaviour was increased. Similar effects were also observed after injection of mCPP into the 3rd ventricle. 3. Co-ordination on a rotating drum of both untrained and trained rats was impaired following mCPP but co-ordination on an elevated bar was not. 4. The hypoactivity induced by mCPP was opposed by three antagonists with high affinity for the 5-hydroxytryptamine (5-HT1C) site; metergoline, mianserin, cyproheptadine and possibly also by a fourth antagonist mesulergine. Metergoline, mianserin and cyproheptadine also opposed the reduction in feeding scores. However, neither effect of mCPP was antagonized by the 5-HT2-receptor antagonists ketanserin or ritanserin, the 5-HT3-receptor antagonist ICS 205-930, the 5-HT1A and 5-HT1B-receptor antagonists (-)-pindolol, (-)-propranolol and (+/-)-cyanopindolol or the 5-HT1A-, 5-HT2- and dopamine receptor antagonist spiperone. The specific alpha 2-adrenoceptor antagonist idazoxan was also without effect. 5. Hypoactivity induced by TFMPP was similarly antagonized by mianserin but unaffected by (+/-)-cyanopindolol. 6. These results suggest that the hypoactivity is mediated by central 5-HT1C-receptors and that mCPP and possibly TFMPP may be 5-HT1C-receptor agonists. 7. As mianserin, cyproheptadine and mesulergine in the absence of mCPP did not increase locomotion but increased the number of feeding scores, the activation of 5-HT1C-receptors may be of physiological importance in the control of appetite. The possible relevance of these results to the therapeutic and side-effects of clinically used antidepressants (particularly

  13. Characterization of Peroxisome Proliferator-Activated Receptor a (PPARa) -Independent Effects of PPARa Activators in the Rodent Liver: Di-(2-ethylhexyl) phthalate Also Activates the Constitutive Activated Receptor

    EPA Science Inventory

    Peroxisome proliferator chemicals (PPC) are thought to mediate their effects in rodents on hepatocyte growth and liver cancer through the nuclear receptor peroxisome proliferatoractivated receptor alpha (PPARa). Recent studies indicate that one such PPC, the plasticizer di2- et...

  14. Current in vitro high throughput screening approaches to assess nuclear receptor activation.

    PubMed

    Raucy, Judy L; Lasker, Jerome M

    2010-11-01

    The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening. PMID:21189134

  15. Five Layers of Receptor Signaling in γδ T-Cell Differentiation and Activation

    PubMed Central

    Ribeiro, Sérgio T.; Ribot, Julie C.; Silva-Santos, Bruno

    2015-01-01

    The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy. PMID:25674089

  16. Myelin-Derived Lipids Modulate Macrophage Activity by Liver X Receptor Activation

    PubMed Central

    Huynh-Thu, Vân Anh; Irrthum, Alexandre; Smeets, Hubert J. M.; Gustafsson, Jan-Åke; Steffensen, Knut R.; Mulder, Monique; Stinissen, Piet; Hellings, Niels; Hendriks, Jerome J. A.

    2012-01-01

    Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. PMID:22984598

  17. Myelin-derived lipids modulate macrophage activity by liver X receptor activation.

    PubMed

    Bogie, Jeroen F J; Timmermans, Silke; Huynh-Thu, Vân Anh; Irrthum, Alexandre; Smeets, Hubert J M; Gustafsson, Jan-Åke; Steffensen, Knut R; Mulder, Monique; Stinissen, Piet; Hellings, Niels; Hendriks, Jerome J A

    2012-01-01

    Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. PMID:22984598

  18. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

    PubMed Central

    Neuman, E; Ladha, M H; Lin, N; Upton, T M; Miller, S J; DiRenzo, J; Pestell, R G; Hinds, P W; Dowdy, S F; Brown, M; Ewen, M E

    1997-01-01

    Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor. PMID:9271411

  19. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  20. Antileishmanial Activity of the Estrogen Receptor Modulator Raloxifene

    PubMed Central

    Reimão, Juliana Q.; Miguel, Danilo C.; Taniwaki, Noemi N.; Trinconi, Cristiana T.; Yokoyama-Yasunaka, Jenicer K. U.; Uliana, Silvia R. B.

    2014-01-01

    Background The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis. Methodology/Principal Findings Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 µM against promastigotes and from 8.8 to 16.2 µM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis – infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden. Conclusions/Significance The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death. PMID:24810565

  1. Topical Peroxisome Proliferator Activated Receptor Activators Accelerate Postnatal Stratum Corneum Acidification

    PubMed Central

    Fluhr, Joachim W.; Man, Mao-Qiang; Hachem, Jean-Pierre; Crumrine, Debra; Mauro, Theodora M.; Elias, Peter M.; Feingold, Kenneth R.

    2015-01-01

    Previous studies have shown that pH declines from between 6 and 7 at birth to adult levels (pH 5.0–5.5) over 5–6 days in neonatal rat stratum corneum (SC). As a result, at birth, neonatal epidermis displays decreased permeability barrier homeostasis and SC integrity, improving days 5–6. We determined here whether peroxisome proliferator-activated receptor (PPAR) activators accelerate postnatal SC acidification. Topical treatment with two different PPARα activators, clofibrate and WY14643, accelerated the postnatal decline in SC surface pH, whereas treatment with PPARγ activators did not and a PPARβ/δ activator had only a modest effect. Treatment with clofibrate significantly accelerated normalization of barrier function. The morphological basis for the improvement in barrier function in PPARα-treated animals includes accelerated secretion of lamellar bodies and enhanced, postsecretory processing of secreted lamellar body contents into mature lamellar membranes. Activity of β-glucocerebrosidase increased after PPARα-activator treatment. PPARα activator also improved SC integrity, which correlated with an increase in corneodesmosome density and increased desmoglein-1 content, with a decline in serine protease activity. Topical treatment of newborn animals with a PPARα activator increased secretory phospholipase A2 activity, which likely accounts for accelerated SC acidification. Thus, PPARα activators accelerate neonatal SC acidification, in parallel with improved permeability homeostasis and SC integrity/cohesion. Hence, PPARα activators might be useful to prevent or treat certain common neonatal dermatoses. PMID:18704104

  2. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Perception of pathogen-associated molecular patterns (PAMPs) by surface-localised pattern-recognition receptors (PRRs) is a key component of plant innate immunity. Most known plant PRRs are receptor kinases and initiation of PAMP-triggered immunity (PTI) signalling requires phosphorylation of the PR...

  3. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    PubMed

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  4. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a

    PubMed Central

    Seredynski, Aurore L.; Balthazart, Jacques; Ball, Gregory F.

    2015-01-01

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β