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Sample records for acetylcholine receptor achieved

  1. Immunisation with Torpedo acetylcholine receptor.

    Elfman, L

    1984-01-01

    Acetylcholine mediates the transfer of information between neurons in the electric organ of, for example, Torpedo as well as in vertebrate skeletal muscle. The nicotinic acetylcholine receptor complex translates the binding of acetylcholine into ion permeability changes. This leads to an action potential in the muscle fibre. The nicotinic acetylcholine receptor protein has been purified from Torpedo by use of affinity chromatography. The receptor is an intrinsic membrane glycoprotein composed of five polypeptide chains. When various animals are immunised with the receptor they demonstrate clinical signs of severe muscle weakness coincident with high antibody titres in their sera. The symptoms resemble those found in the autoimmune neuromuscular disease myasthenia gravis in humans. This animal model has constituted a unique model for studying autoimmune diseases. This paper reviews some of the work using Torpedo acetylcholine receptor in order to increase the understanding of the motor nervous system function and myasthenia gravis. It is now known that the nicotinic acetylcholine receptor protein is the antigen involved in myasthenia gravis. The mechanism of immune damage involves a direct block of the receptor function. This depends on the presence of antibodies which crosslink the postsynaptic receptors leading to their degradation. The questions to be answered in the future are; (a) what initiates or triggers the autoimmune response, (b) how do the antibodies cause the symptoms--is there a steric hindrance of the interaction of acetylcholine and the receptor, (c) why is there not a strict relationship between antibody titre and severity of symptoms, and (d) why are some muscles affected and other spared? With help of the experimental model, answers to these questions may result in improved strategies for the treatment of the autoimmune disease myasthenia gravis. PMID:6097937

  2. Tubular crystals of acetylcholine receptor

    1984-01-01

    Well-ordered tubular crystals of acetylcholine receptor were obtained from suspensions of Torpedo marmorata receptor-rich vesicles. They are composed of pairs of oppositely oriented molecules arranged on the surface lattice with the symmetry of the plane group p2 (average unit cell dimensions: a = 90 A, b = 162 A, gamma = 117 degrees). The receptor in this lattice has an asymmetric distribution of mass around its perimeter, yet a regular pentagonal shape; thus its five transmembrane subunits ...

  3. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Esam E. El-Fakahany

    2010-08-01

    Full Text Available An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity or decrease (negative cooperativity in the binding or action of an orthosteric agonist (e.g., acetylcholine. Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer’s disease and other disorders involving impaired cognitive function.

  4. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Jakubík, Jan; El-Fakahany, E. E.

    2010-01-01

    Roč. 3, č. 9 (2010), s. 2838-2860. ISSN 1424-8247 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : muscarinic acetylcholine receptors * allosteric modulation * Alzheimer ´s disease Subject RIV: CE - Biochemistry

  5. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    Rozman, Klara Bulc; Araoz, Romulo; Sepcić, Kristina; Molgo, Jordi; Suput, Dusan

    2010-09-01

    Nicotinic acetylcholine receptors are implicated in different nervous system-related disorders, and their modulation could improve existing therapy of these diseases. Parazoanthoxanthin A (ParaA) is a fluorescent pigment of the group of zoanthoxanthins. Since it is a potent acetylcholinesterase inhibitor, it may also bind to nicotinic acetylcholine receptors (nAChRs). For this reason its effect on Torpedo nAChR (alpha1(2)betagammadelta) transplanted to Xenopus laevis oocytes was evaluated, using the voltage-clamp technique. ParaA dose-dependently reduced the acetylcholine-induced currents. This effect was fully reversible only at lower concentrations. ParaA also reduced the Hill coefficient and the time to peak current, indicating a channel blocking mode of action. On the other hand, the combined effect of ParaA and d-tubocurarine (d-TC) on acetylcholine-induced currents exhibited only partial additivity, assuming a competitive mode of action of ParaA on nAChR. These results indicate a dual mode of action of ParaA on the Torpedo AChR. PMID:20230806

  6. Expression and function of nicotinic acetylcholine receptors in stem cells

    Carlos M. Carballosa

    2016-07-01

    Full Text Available Nicotinic acetylcholine receptors are prototypical ligand gated ion channels typically found in muscular and neuronal tissues. Functional nicotinic acetylcholine receptors, however, have also recently been identified on other cell types, including stem cells. Activation of these receptors by the binding of agonists like choline, acetylcholine, or nicotine has been implicated in many cellular changes. In regards to stem cell function, nicotinic acetylcholine receptor activation leads to changes in stem cell proliferation, migration and differentiation potential. In this review we summarize the expression and function of known nicotinic acetylcholine receptors in different classes of stem cells including: pluripotent stem cells, mesenchymal stem cells, periodontal ligament derived stem cells, and neural progenitor cells and discuss the potential downstream effects of receptor activation on stem cell function.

  7. The α7 nicotinic acetylcholine receptor complex

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds and...... compounds in vivo is highly dependent on α7 nAChR-interacting proteins, such as RIC-3 and lynx1, which modulate expression and function of the receptor. These regulatory proteins are often not expressed in in vitro models used to study α7 nAChR function, and it is not known to what extent they are involved...... in diseases such as schizophrenia and Alzheimer's disease. Furthermore, α7 nAChR agonists and allosteric modulators differentially alter expression and functionality of the α7 nAChR with repeated administration, which suggests that there may be fundamentally different outcomes of long...

  8. Acetylcholine affects osteocytic MLO-Y4 cells via acetylcholine receptors.

    Ma, Yuanyuan; Li, Xianxian; Fu, Jing; Li, Yue; Gao, Li; Yang, Ling; Zhang, Ping; Shen, Jiefei; Wang, Hang

    2014-03-25

    The identification of the neuronal control of bone remodeling has become one of the many significant recent advances in bone biology. Cholinergic activity has recently been shown to favor bone mass accrual by complex cellular regulatory networks. Here, we identified the gene expression of the muscarinic and nicotinic acetylcholine receptors (m- and nAChRs) in mice tibia tissue and in osteocytic MLO-Y4 cells. Acetylcholine, which is a classical neurotransmitter and an osteo-neuromediator, not only influences the mRNA expression of the AChR subunits but also significantly induces the proliferation and viability of osteocytes. Moreover, acetylcholine treatment caused the reciprocal regulation of RANKL and OPG mRNA expression, which resulted in a significant increase in the mRNA ratio of RANKL:OPG in osteocytes via acetylcholine receptors. The expression of neuropeptide Y and reelin, which are two neurogenic markers, was also modulated by acetylcholine via m- and nAChRs in MLO-Y4 cells. These results indicated that osteocytic acetylcholine receptors might be a new valuable mediator for cell functions and even for bone remodeling. PMID:24508663

  9. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine Binding Protein as a Structural Surrogate

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette; Balle, Thomas

    2015-01-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for development of drugs a...

  10. Nicotinic acetylcholine receptors mediate lung cancer growth

    PaulDGardner

    2013-09-01

    Full Text Available Ion channels modulate ion flux across cell membranes, activate signal transduction pathways, and influence cellular transport – vital biological functions that are inexorably linked to cellular processes that go awry during carcinogenesis. Indeed, deregulation of ion channel function has been implicated in cancer-related phenomena such as unrestrained cell proliferation and apoptotic evasion. As the prototype for ligand-gated ion channels, nicotinic acetylcholine receptors (nAChRs have been extensively studied in the context of neuronal cells but accumulating evidence also indicate a role for nAChRs in carcinogenesis. Recently, variants in the nAChR genes CHRNA3, CHRNA5, and CHRNB4 have been implicated in nicotine dependence and lung cancer susceptibility. Here, we silenced the expression of these three genes to investigate their function in lung cancer. We show that these genes are necessary for the viability of small cell lung carcinomas (SCLC, the most aggressive type of lung cancer. Furthermore, we show that nicotine promotes SCLC cell viability whereas an α3β4-selective antagonist, α-conotoxin AuIB, inhibits it. Our findings posit a mechanism whereby signaling via α3/α5/β4-containing nAChRs promotes lung carcinogenesis.

  11. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  12. Functional partial agonism at cloned human muscarinic acetylcholine receptors

    Bräuner-Osborne, Hans; Ebert, B; Brann, M R;

    1996-01-01

    of maximal response, depending on the molar ratio of agonist and antagonist used. Using recombinant human muscarinic acetylcholine receptors (m1 and m5) and the functional assay, receptor selection and amplification technology (R-SAT), we have now shown that co-administration of the full agonist...

  13. Positive cooperativity of acetylcholine and other agonists with allosteric ligands on muscarinic acetylcholine receptors.

    Jakubík, J; Bacáková, L; El-Fakahany, E E; Tucek, S

    1997-07-01

    It is well known that allosteric modulators of muscarinic acetylcholine receptors can both diminish and increase the affinity of receptors for their antagonists. We investigated whether the allosteric modulators can also increase the affinity of receptors for their agonists. Twelve agonists and five allosteric modulators were tested in experiments on membranes of CHO cells that had been stably transfected with genes for the M1-M4 receptor subtypes. Allosterically induced changes in the affinities for agonists were computed from changes in the ability of a fixed concentration of each agonist to compete with [3H]N-methylscopolamine for the binding to the receptors in the absence and the presence of varying concentrations of allosteric modulators. The effects of allosteric modulators varied greatly depending on the agonists and the subtypes of receptors. The affinity for acetylcholine was augmented by (-)-eburnamonine on the M2 and M4 receptors and by brucine on the M1 and M3 receptors. Brucine also enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pilocarpine, 3-(3-pentylthio-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1- methylpyridine (pentylthio-TZTP), oxotremorine-M, and McN-A-343 on the M1, M3, and M4 receptors, for pentylthio-TZTP on the M2 receptors, and for arecoline on the M3 receptors. (-)-Eburnamonine enhanced the affinities for carbachol, bethanechol, furmethide, methylfurmethide, pentylthio-TZTP, pilocarpine, oxotremorine and oxotremorine-M on the M2 receptors and for pilocarpine on the M4 receptors. Vincamine, strychnine, and alcuronium displayed fewer positive allosteric interactions with the agonists, but each allosteric modulator displayed positive cooperativity with at least one agonist on at least one muscarinic receptor subtype. The highest degrees of positive cooperativity were observed between (-)-eburnamonine and pilocarpine and (-)-eburnamonine and oxotremorine-M on the M2 receptors (25- and 7-fold increases in

  14. Structural Studies of Nicotinic Acetylcholine Receptors: Using Acetylcholine-Binding Protein as a Structural Surrogate.

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette S; Balle, Thomas

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the pentameric ligand-gated ion channel superfamily that play important roles in the control of neurotransmitter release in the central and peripheral nervous system. These receptors are important therapeutic targets for the development of drugs against a number of mental health disorders and for marketed smoking cessation aids. Unfortunately, drug discovery has been hampered by difficulties in obtaining sufficiently selective compounds. Together with functional complexity of the receptors, this has made it difficult to obtain drugs with sufficiently high-target to off-target affinity ratios. The recent and ongoing progress in structural studies holds promise to help understand structure-function relationships of nAChR drugs at the atomic level. This will undoubtedly lead to the design of more efficient drugs with fewer side effects. As a high-resolution structure of a nAChR is yet to be determined, structural studies are to a large extent based on acetylcholine-binding proteins (AChBPs) that despite low overall sequence identity display a high degree of conservation of overall structure and amino acids at the ligand-binding site. Further, AChBPs reproduce relative binding affinities of ligands at nAChRs. Over the past decade, AChBPs have been used extensively as models for nAChRs and have aided the understanding of drug receptor interactions at nAChRs significantly. PMID:26572235

  15. Localization of muscarinic acetylcholine receptor in plant guard cells

    2001-01-01

    Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. In Vicia faba L. and Pisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane of Vicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.

  16. Structural model of nicotinic acetylcholine receptor isotypes bound to acetylcholine and nicotine

    Abagyan Ruben

    2002-01-01

    Full Text Available Abstract Background Nicotine is a psychoactive drug presenting a diverse array of biological activities, some positive, such as enhancement of cognitive performances, others negative, such as addiction liability. Ligands that discriminate between the different isotypes of nicotinic acetylcholine receptors (nAChRs could present improved pharmacology and toxicity profile. Results Based on the recent crystal structure of a soluble acetylcholine binding protein from snails, we have built atomic models of acetylcholine and nicotine bound to the pocket of four different human nAChR subtypes. The structures of the docked ligands correlate with available biochemical data, and reveal that the determinants for isotype selectivity are relying essentially on four residues, providing diversity of the ligand binding pocket both in terms of Van der Waals boundary, and electrostatic potential. We used our models to screen in silico a large compound database and identify a new ligand candidate that could display subtype selectivity. Conclusion The nAChR-agonist models should be useful for the design of nAChR agonists with diverse specificity profiles.

  17. Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

    Alexandra eAcevedo-Rodriguez; Lifen eZhang; Fuwen eZhou; Suzhen eGong; Howard eGu; Mariella eDe Biasi; Fu-Ming eZhou; Dani, John A.

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) potently regulate dopamine (DA) release in the striatum and alter cocaine’s ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit...

  18. Threonine in the selectivity filter of the acetylcholine receptor channel.

    Villarroel, A.; Sakmann, B

    1992-01-01

    The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild...

  19. Subtype Differences in Pre-Coupling of Muscarinic Acetylcholine Receptors

    Jakubík, Jan; Janíčková, Helena; Randáková, Alena; El-Fakahany, E. E.; Doležal, Vladimír

    2011-01-01

    Roč. 6, č. 11 (2011), e27732. E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GA305/09/0681; GA AV ČR(CZ) IAA500110703; GA MŠk(CZ) LC554 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine muscarinic receptors * G proteins * subtype differences Subject RIV: ED - Physiology Impact factor: 4.092, year: 2011

  20. A new family of insect muscarinic acetylcholine receptors.

    Xia, R-Y; Li, M-Q; Wu, Y-S; Qi, Y-X; Ye, G-Y; Huang, J

    2016-08-01

    Most currently used insecticides are neurotoxic chemicals that target a limited number of sites and insect cholinergic neurotransmission is the major target. A potential target for insecticide development is the muscarinic acetylcholine receptor (mAChR), which is a metabotropic G-protein-coupled receptor. Insects have A- and B-type mAChRs and the five mammalian mAChRs are close to the A-type. We isolated a cDNA (CG12796) from the fruit fly, Drosophila melanogaster. After heterologous expression in Chinese hamster ovary K1 cells, CG12796 could be activated by acetylcholine [EC50 (half maximal effective concentration), 73 nM] and the mAChR agonist oxotremorine M (EC50 , 48.2 nM) to increase intracellular Ca(2+) levels. Thus, the new mAChR is coupled to Gq/11 but not Gs and Gi/o . The classical mAChR antagonists atropine and scopolamine N-butylbromide at 100 μM completely blocked the acetylcholine-induced responses. The orthologues of CG12796 can also be found in the genomes of other insects, but not in the genomes of the honeybee or parasitoid wasps. Knockdown of CG12796 in the central nervous system had no effect on male courtship behaviours. We suggest that CG12796 represents the first recognized member of a novel mAChR class. PMID:27003873

  1. Gold nanoparticle–choline complexes can block nicotinic acetylcholine receptors

    Chur Chin

    2010-04-01

    Full Text Available Chur Chin1, In Kyeom Kim2, Dong Yoon Lim3, Ki Suk Kim4, Hyang Ae Lee4, Eun Joo Kim41Department of Pediatrics, Fatima Hospital, Daegu, Korea; 2Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Korea; 3Department of Pharmacology, School of Medicine, Chosun University, Gwangju, Korea; 4Korea Institute of Toxicology, Daejeon, KoreaAbstract: We identified a novel class of direct ion-channel blockers of ligand-gated ion channels called the gold nanoparticle–choline complex. Negatively charged gold nanoparticles (1.4 nm block ion pores by binding to the sulfur group of the cysteine loop of nicotinic acetylcholine receptors (nAChRs, and currents evoked by acetylcholine (Ach can break these bonds. The current evoked by ACh in nAChRs was blocked directly in ion pores by the gold nanoparticle–choline complex. In adrenal-gland perfusion studies, the complex also blocked nAChRs by diminishing catecholamine release by about 75%. An in vivo study showed muscle relaxation in rats after injection of the complex. These results will foster the application of gold nanoparticles as a direct ion-channel blocker. Keywords: negatively charged gold nanoparticle, choline, gold–sulfur bond, nicotinic acetylcholine receptor, direct ion-channel blocker

  2. Molecular alteration of a muscarinic acetylcholine receptor system during synaptogenesis

    Biochemical properties of the muscarinic acetylcholine receptor system of the avian retina were found to change during the period when synapses form in ovo. Comparison of ligand binding to membranes obtained before and after synaptogenesis showed a significant increase in the affinity, but not proportion, of the high affinity agonist-binding state. There was no change in receptor sensitivity to antagonists during this period. Pirenzepine binding, which can discriminate muscarinic receptor subtypes, showed the presence of a single population of low affinity sites (M2) before and after synaptogenesis. The change in agonist binding was not due to the late development of receptor function. However, detergent-solubilization of membranes eliminated differences in agonist binding between receptors from embryos and hatched chicks, suggesting a developmental change in interactions of the receptor with functionally related membrane components. A possible basis for altered interactions was obtained from isoelectric point data showing that the muscarinic receptor population underwent a transition from a predominantly low pI form (4.25) in 13 day embryos to a predominantly high pI form (4.50) in newly hatched chicks. The possibility that biochemical changes in the muscarinic receptor play a role in differentiation of the system by controlling receptor position on the surface of nerve cells is discussed

  3. Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

    Hugh J Freeman; Helen R Gillett; Peter M Gillett; Joel Oger

    2009-01-01

    Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a glutenfree diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.

  4. Effect of organophosphorus insecticides on phosphorylation of the M2 muscarinic acetylcholine receptor

    Shuyin Li; Liming Zou; Carry Pope

    2008-01-01

    BACKGROUND: Organophosphorus insecticides may promote the accumulation of acetylcholine at synapses and the neuromuscular junction by inhibiting acetylcholinesterase activity to cause disturbance of neural signal conduction and induce a toxic reaction. Organophosphorus insecticides may act on M2 muscarinic acetylcholine receptors, whose combination with G proteins is regulated by phosphorylation of G protein-coupled receptor kinase 2.OBJECTIVE: To investigate the effects of organophosphorus insecticides on the phosphorylation of G protein-coupled receptor kinase 2-mediated M2 muscarinic acetylcholine receptors and to reveal other possible actions of organophosphorus insecticides.DESIGN, TIME AND SETTING: An observational study, which was performed in the Central Laboratory of Shenyang Medical College, and Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University from June 2002 to December 2004.METHODS: The M2 muscarinic acetylcholine receptor was extracted and purified from pig brain using affinity chromatography. Subsequently, the purified M2 muscarinic acetylcholine receptor, G protein-coupled receptor kinase 2, and [OP32] ATP were incubated with different concentrations of paraoxon and chlorpyrifos oxon together. The mixture then underwent polyacrylamide gel electrophoresis, and the gel film was dried and radioactively autographed to detect phosphorylation of the M2 muscarinic acetylcholine receptor. Finally, the radio-labeled phosphorylated M2 receptor protein band was excised for counting with an isotope liquid scintillation counter.MAIN OUTCOME MEASURES: Effects of chlorpyrifos oxon, paraoxon, chlorpyrifos, and parathion in different concentrations on the phosphorylation of the M2 muscarinic acetylcholine receptor; effects of chlorpyrifos oxon on the phosphorylation of the adrenergic receptor.CONCLUSION: Different kinds of organophosphorus insecticides have different effects on the phosphorylation of the G protein

  5. Schizophrenia and the alpha7 nicotinic acetylcholine receptor.

    Martin, Laura F; Freedman, Robert

    2007-01-01

    In addition to the devastating symptoms of psychosis, many people with schizophrenia also suffer from cognitive impairment. These cognitive symptoms lead to marked dysfunction and can impact employability, treatment adherence, and social skills. Deficits in P50 auditory gating are associated with attentional impairment and may contribute to cognitive symptoms and perceptual disturbances. This nicotinic cholinergic-mediated inhibitory process represents a potential new target for therapeutic intervention in schizophrenia. This chapter will review evidence implicating the nicotinic cholinergic, and specifically, the alpha7 nicotinic receptor system in the pathology of schizophrenia. Impaired auditory sensory gating has been linked to the alpha7 nicotinic receptor gene on the chromosome 15q14 locus. A majority of persons with schizophrenia are heavy smokers. Although nicotine can acutely reverse diminished auditory sensory gating in people with schizophrenia, this effect is lost on a chronic basis due to receptor desensitization. The alpha7 nicotinic agonist 3-(2,4 dimethoxy)benzylidene-anabaseine (DMXBA) can also enhance auditory sensory gating in animal models. DMXBA is well tolerated in humans and a new study in persons with schizophrenia has found that DMXBA enhances both P50 auditory gating and cognition. alpha7 Nicotinic acetylcholine receptor agonists appear to be viable candidates for the treatment of cognitive disturbances in schizophrenia. PMID:17349863

  6. Valence of acetylcholine-receptor-antibody-titers in myasthenia gravis

    In a retrospective study in 47 patients with myasthenia gravis acetylcholine-receptor-antibody-titers (AChR-AB) were correlated with the severity of the disease. In 18 patients the course of titers was studied and two groups of patients could be differentiated: patients with relative constant and patients with fluctuating titers. Age, age of begin of myasthenia and sex did not influence the titers. Also the duration of the disease and the severity of symptoms did not influence the level of AChR-AB-titers. In this retrospective study the influence of immunsuppressive therapy on the intra-individual course of AB-titers and their correlation with the clinical symptoms could not be judged. Measurement of AChR-AB is of value for the diagnosis of myasthenia gravis and important for judging the clinical course and the effect of therapy. (Author)

  7. Two types of muscarinic acetylcholine receptors in Drosophila and other arthropods

    Collin, Caitlin Alexis; Hauser, Frank; Gonzalez de Valdivia, Ernesto I; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M.M.; Khan, Zaid; Hansen, Niels Ø.; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin Robin; Grimmelikhuijzen, Cornelis

    2013-01-01

    Muscarinic acetylcholine receptors (mAChRs) play a central role in the mammalian nervous system. These receptors are G protein-coupled receptors (GPCRs), which are activated by the agonists acetylcholine and muscarine, and blocked by a variety of antagonists. Mammals have five mAChRs (m1-m5). In ...... (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs....

  8. Conformationally restrained carbamoylcholine homologues. Synthesis, pharmacology at neuronal nicotinic acetylcholine receptors and biostructural considerations

    de la Fuente Revenga, M; Balle, Thomas; Jensen, Anders A.; Frølund, Bente

    2015-01-01

    Exploration of small selective ligands for the nicotinic acetylcholine receptors (nAChRs) based on acetylcholine (ACh) has led to the development of potent agonists with clear preference for the α4β2 nAChR, the most prevalent nAChR subtype in the central nervous system. In this work we present the...

  9. Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

    The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with (3H)choline. In the presence of H1 and H2 blockade, histamine and (R)-α-methylhistamine inhibited the electrically-evoked acetylcholine release, being (R)-α-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-α-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors

  10. Evaluation of PET Radioligands for the neuronal nicotinic acetylcholine receptor

    Full text: A-186253.1, a compound made by Abbott laboratories, was labelled with carbon-11 and evaluated as a PET ligand for the neuronal nicotinic acetylcholine receptor (nAChR). The compound was labelled with C-11 by methylation with 11C-MeI of the desmethyl precursor A-183828.1. The affinity of A-186253.1 for the α4β2 and the α7 subtype of the nAChR was determined in displacement studies. PET-studies were performed in rats and pigs Inhibitory constants (Ki) versus cytsine were 461 ± 99 pM for A-186253.1 and versus α-Bungarotoxin >100 μM. which means a very high selectivity for the α4β2-receptor (>227,000). Highest uptake of [11C]-A-186253.1 was observed in the thalamus where an increase in radiotracer uptake was seen until 45 min p.i.. Thereafter, the radiotracer concentration remained constant until the end of the scan indicating slow washout of [11C]-A-186253.1. Application of cold A-186253.1 (0.5 mg/kg) 40 min p.i. resulted in a decrease in radiotracer concentration in the thalamus and the cortex indicating displacement of [11C]-A-186253.1. Blockade studies with cytisine (0.5 mg/kg), a selective ligand for the α4β2 nicotinic receptor, showed just a slight reduction of the radioligand uptake in the thalamus and in the cortex whereas the blockade with cold A-186253.1 (1 mg/kg) resulted in a 50 % reduction. These results suggest, that 50 % of the [11C]-A-186253.1 in the brain corresponds to specifically bound radioligand, but not to the α4β2 subtype of the nicotinic receptor. (author)

  11. Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

    O'Reilly, Clare; Pette, Dirk; Ohlendieck, Kay

    2003-01-10

    Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts. PMID:12504123

  12. Effects of two oxadiazolidinones on cholinesterases and acetylcholine receptors

    Inhibition of acetylcholinesterase (AChE) and butyryl cholinesterase (BuChE) by 3-(2,3-dihydro-2,2-dimethyl-benzofuran-'7-yl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (DBOX) and 3-(2-methoxyphenyl)-5-methoxy-1,3,4-oxadiazol-2(3H)-one (MPOX) was measured by the Ellmann spectrophotometric method. Inhibition was quasi first order and irreversible. DBOX was 2-3 orders of magnitude more potent than MPOX. Housefly brain AChE and horse serum BuChE were more sensitive than AChEs of red blood cells or eel and Torpedo electric organs. It is suggested that the nonesteratic oxadiazolidinones are activated to carbanillates on the surface of the enzyme and produce a carbanillated enzyme which ages rapidly. Carbamate anticholinesterases protected AChE against carbanillation as they did against phosphorylation. At higher concentrations, the two oxadiazolidinones also affected binding of [125I] α bungarotoxin and [3H]perhydrohistrionicotoxin to Torpedo nicotinic acetylcholine receptors, but did not affect binding of [3H]quinuclidinyl benzilate to rat brain muscarinic receptors

  13. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

    Bertrand, Daniel; Lee, Chih-Hung L; Flood, Dorothy; Marger, Fabrice; Donnelly-Roberts, Diana

    2015-10-01

    Progress in the fields of neuroscience and molecular biology has identified the forebrain cholinergic system as being important in many higher order brain functions. Further analysis of the genes encoding the nicotinic acetylcholine receptors (nAChRs) has highlighted, in particular, the role of α7 nAChRs in these higher order brain functions as evidenced by their peculiar physiologic and pharmacological properties. As this receptor has gained the attention of scientists from academia and industry, our knowledge of its roles in various brain and bodily functions has increased immensely. We have also seen the development of small molecules that have further refined our understanding of the roles of α7 nAChRs, and these molecules have begun to be tested in clinical trials for several indications. Although a large body of data has confirmed a role of α7 nAChRs in cognition, the translation of small molecules affecting α7 nAChRs into therapeutics has to date only progressed to the stage of testing in clinical trials. Notably, however, most recent human genetic and biochemical studies are further underscoring the crucial role of α7 nAChRs and associated genes in multiple organ systems and disease states. The aim of this review is to discuss our current knowledge of α7 nAChRs and their relevance as a target in specific functional systems and disease states. PMID:26419447

  14. Expression of the α7 nicotinic acetylcholine receptor in human lung cells

    Schuller Hildegard M; Dhar Madhu; Plummer Howard K

    2005-01-01

    Abstract Background We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was t...

  15. VISUALIZATION OF CHOLINOCEPTIVE NEURONS IN THE RAT NEOCORTEX - COLOCALIZATION OF MUSCARINIC AND NICOTINIC ACETYLCHOLINE-RECEPTORS

    VANDERZEE, EA; STREEFLAND, C; STROSBERG, AD; SCHRODER, H; LUITEN, PGM

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluores

  16. Visualization of cholinoceptive neurons in the rat neocortex : colocalization of muscarinic and nicotinic acetylcholine receptors

    Zee, E.A. van der; Streefland, C.; Strosberg, A.D.; Schröder, H.; Luiten, P.G.M.

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluores

  17. Nicotinic Acetylcholine Receptor (nAChR Dependent Chorda Tympani Taste Nerve Responses to Nicotine, Ethanol and Acetylcholine.

    Zuo Jun Ren

    Full Text Available Nicotine elicits bitter taste by activating TRPM5-dependent and TRPM5-independent but neuronal nAChR-dependent pathways. The nAChRs represent common targets at which acetylcholine, nicotine and ethanol functionally interact in the central nervous system. Here, we investigated if the nAChRs also represent a common pathway through which the bitter taste of nicotine, ethanol and acetylcholine is transduced. To this end, chorda tympani (CT taste nerve responses were monitored in rats, wild-type mice and TRPM5 knockout (KO mice following lingual stimulation with nicotine free base, ethanol, and acetylcholine, in the absence and presence of nAChR agonists and antagonists. The nAChR modulators: mecamylamine, dihydro-β-erythroidine, and CP-601932 (a partial agonist of the α3β4* nAChR, inhibited CT responses to nicotine, ethanol, and acetylcholine. CT responses to nicotine and ethanol were also inhibited by topical lingual application of 8-chlorophenylthio (CPT-cAMP and loading taste cells with [Ca2+]i by topical lingual application of ionomycin + CaCl2. In contrast, CT responses to nicotine were enhanced when TRC [Ca2+]i was reduced by topical lingual application of BAPTA-AM. In patch-clamp experiments, only a subset of isolated rat fungiform taste cells exposed to nicotine responded with an increase in mecamylamine-sensitive inward currents. We conclude that nAChRs expressed in a subset of taste cells serve as common receptors for the detection of the TRPM5-independent bitter taste of nicotine, acetylcholine and ethanol.

  18. Alpha-conotoxins as pharmacological probes of nicotinic acetylcholine receptors

    Layla AZAM; J Michael MCINTOSH

    2009-01-01

    Cysteine-rich peptides from the venom of cone snails (Conus) target a wide variety of different ion channels. One family of conopeptides, the a-conotoxins, specifically target different isoforms of nicotinic acetylcholine receptors (nAChRs) found both in the neuromuscular junction and central nervous system. This family is further divided into subfamilies based on the number of amino acids between cysteine residues. The exquisite subtype selectivity of certain a-conotoxins has been key to the characterization of native nAChR isoforms involved in modulation of neurotransmitter release, the pathophysiol-ogy of Parkinson's disease and nociception. Structure/function characterization of a-conotoxins has led to the development of analogs with improved potency and/or subtype selectivity. Cyclization of the backbone structure and addition of lipo-philic moieties has led to improved stability and bioavailability of a-conotoxins, thus paving the way for orally available therapeutics. The recent advances in phylogeny, exogenomics and molecular modeling promises the discovery of an even greater number of a-conotoxins and analogs with improved selectivity for specific subtypes of nAChRs.

  19. Cocaine Inhibition of Nicotinic Acetylcholine ReceptorsInfluences Dopamine Release

    Alexandra eAcevedo-Rodriguez

    2014-09-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs potently regulate dopamine (DA release in the striatum and alter cocaine’s ability to reinforce behaviors. Since cocaine is a weak nAChR inhibitor, we hypothesized that cocaine may alter DA release by inhibiting the nAChRs in DA terminals in the striatum and thus contribute to cocaine's reinforcing properties primarily associated with the inhibition of DA transporters. We found that biologically relevant concentrations of cocaine can mildly inhibit nAChR-mediated currents in midbrain DA neurons and consequently alter DA release in the dorsal and ventral striatum. At very high concentrations, cocaine also inhibits voltage-gated Na channels in DA neurons. Furthermore, our results show that partial inhibition of nAChRs by cocaine reduces evoked DA release. This diminution of DA release via nAChR inhibition more strongly influences release evoked at low or tonic stimulation frequencies than at higher (phasic stimulation frequencies, particularly in the dorsolateral striatum. This cocaine-induced shift favoring phasic DA release may contribute to the enhanced saliency and motivational value of cocaine-associated memories and behaviors.

  20. Effects of the α subunit on imidacloprid sensitivity of recombinant nicotinic acetylcholine receptors

    Matsuda, K; Buckingham, S D; Freeman, J.C.; Squire, M D; Baylis, H. A.; Sattelle, D B

    1998-01-01

    Imidacloprid is a new insecticide with selective toxicity for insects over vertebrates. Recombinant (α4β2) chicken neuronal nicotinic acetylcholine receptors (AChRs) and a hybrid nicotinic AChR formed by co-expression of a Drosophila melanogaster neuronal α subunit (SAD) with the chicken β2 subunit were heterologously expressed in Xenopus oocytes by nuclear injection of cDNAs. The agonist actions of imidacloprid and other nicotinic AChR ligands ((+)-epibatidine, (−)-nicotine and acetylcholine...

  1. Acetylcholine receptors in dementia and mild cognitive impairment

    Sabri, Osama; Kendziorra, Kai [University of Leipzig, Department of Nuclear Medicine, Leipzig (Germany); Wolf, Henrike; Gertz, Hermann-Josef [University of Leipzig, Department of Psychiatry, Leipzig (Germany); Brust, Peter [Institute of Interdisciplinary Isotope Research, Leipzig (Germany)

    2008-03-15

    To clarify whether changes in the cholinergic transmission occur early in the course of Alzheimer's disease (AD), we carried out positron emission tomography (PET) with the radioligand 2-[{sup 18}F]F-A-85380, which is supposed to be specific for {alpha}4{beta}2 nicotinic acetylcholine receptors (nAChRs). We included patients with moderate to severe AD and patients with amnestic mild cognitive impairment (MCI), presumed to present preclinical AD. Both patients with AD and MCI showed significant reductions in {alpha}4{beta}2 nAChRs in brain regions typically affected by AD pathology. These findings indicate that a reduction in {alpha}4{beta}2 nAChRs occurs during early symptomatic stages of AD. The {alpha}4{beta}2 nAChR availability in these regions correlated with the severity of cognitive impairment, indicating a stage sensitivity of the {alpha}4{beta}2 nAChR status. Together, our results provide evidence for the potential of 2-[{sup 18}]F-A-85380 nAChR PET in the diagnosis of patients at risk for AD. Because of the extraordinary long acquisition time with 2-[{sup 18}F]F-A-85380, we developed the new {alpha}4{beta}2 nAChR-specific radioligands (+)- and (-)-[{sup 18}F]norchloro-fluoro-homoepibatidine (NCFHEB) and evaluated them preclinically. (-)-[{sup 18}F]NCFHEB shows twofold higher brain uptake and significantly shorter acquisition times. Therefore, (-)-[{sup 18}F]NCFHEB should be a suitable radioligand for larger clinical investigations. (orig.)

  2. Acetylcholine receptors in dementia and mild cognitive impairment

    To clarify whether changes in the cholinergic transmission occur early in the course of Alzheimer's disease (AD), we carried out positron emission tomography (PET) with the radioligand 2-[18F]F-A-85380, which is supposed to be specific for α4β2 nicotinic acetylcholine receptors (nAChRs). We included patients with moderate to severe AD and patients with amnestic mild cognitive impairment (MCI), presumed to present preclinical AD. Both patients with AD and MCI showed significant reductions in α4β2 nAChRs in brain regions typically affected by AD pathology. These findings indicate that a reduction in α4β2 nAChRs occurs during early symptomatic stages of AD. The α4β2 nAChR availability in these regions correlated with the severity of cognitive impairment, indicating a stage sensitivity of the α4β2 nAChR status. Together, our results provide evidence for the potential of 2-[18]F-A-85380 nAChR PET in the diagnosis of patients at risk for AD. Because of the extraordinary long acquisition time with 2-[18F]F-A-85380, we developed the new α4β2 nAChR-specific radioligands (+)- and (-)-[18F]norchloro-fluoro-homoepibatidine (NCFHEB) and evaluated them preclinically. (-)-[18F]NCFHEB shows twofold higher brain uptake and significantly shorter acquisition times. Therefore, (-)-[18F]NCFHEB should be a suitable radioligand for larger clinical investigations. (orig.)

  3. Enhanced self-administration of alcohol in muscarinic acetylcholine M4 receptor knockout mice

    de la Cour, Cecilie; Sørensen, Gunnar; Wörtwein, Gitta;

    2015-01-01

    Modulation of cholinergic neurotransmission via nicotinic acetylcholine receptors is known to alter alcohol-drinking behavior. It is not known if muscarinic acetylcholine receptor subtypes have similar effects. The muscarinic M4 receptor is highly expressed in the brain reinforcement system and i......4+/+ littermates. The highest alcohol concentration used (10%) did not immediately result in divergent drinking patterns, but after 4 weeks of 10% alcohol self-administration, baseline levels as well as a pattern of M4-/- mice consuming more alcohol than their M4+/+ controls were re...... as a potential target for pharmacological (positive allosteric modulators or future agonists) treatment of alcohol use disorders....

  4. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells

    L.F.S. Sampaio

    2005-04-01

    Full Text Available The influence of melatonin on the developmental pattern of functional nicotinic acetylcholine receptors was investigated in embryonic 8-day-old chick retinal cells in culture. The functional response to acetylcholine was measured in cultured retina cells by microphysiometry. The maximal functional response to acetylcholine increased 2.7 times between the 4th and 5th day in vitro (DIV4, DIV5, while the Bmax value for [125I]-alpha-bungarotoxin was reduced. Despite the presence of alpha8-like immunoreactivity at DIV4, functional responses mediated by alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors were observed only at DIV5. Mecamylamine (100 µM was essentially without effect at DIV4 and DIV5, while dihydro-ß-erythroidine (10-100 µM blocked the response to acetylcholine (3.0 nM-2.0 µM only at DIV4, with no effect at DIV5. Inhibition of melatonin receptors with the antagonist luzindole, or melatonin synthesis by stimulation of D4 dopamine receptors blocked the appearance of the alpha-bungarotoxin-sensitive response at DIV5. Therefore, alpha-bungarotoxin-sensitive receptors were expressed in retinal cells as early as at DIV4, but they reacted to acetylcholine only after DIV5. The development of an alpha-bungarotoxin-sensitive response is dependent on the production of melatonin by the retinal culture. Melatonin, which is produced in a tonic manner by this culture, and is a key hormone in the temporal organization of vertebrates, also potentiates responses mediated by alpha-bungarotoxin-sensitive receptors in rat vas deferens and cerebellum. This common pattern of action on different cell models that express alpha-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.

  5. Visualization of cholinoceptive neurons in the rat neocortex: colocalization of muscarinic and nicotinic acetylcholine receptors

    Zee, E.A. van der; Streefland, C.; Strosberg, A D; Schröder, H.; Luiten, P.G.M.

    1992-01-01

    The present investigation analyzes the cellular distribution of muscarinic and nicotinic acetylcholine receptors in rat neocortex, by use of monoclonal antibodies raised against purified receptor proteins. The degree of colocalization of both types of receptors was determined by way of immunofluorescent double-labeling techniques. For both classes of receptors, pyramidal and nonpyramidal cells were found immunostained and an identical laminar distribution pattern of immunopositive neurons in ...

  6. Theoretical investigation of interaction between the set of ligands and α7 nicotinic acetylcholine receptor

    Glukhova, O. E.; Prytkova, T. R.; Shmygin, D. S.

    2016-03-01

    Nicotinic acetylcholine receptors (nAChRs) are neuron receptor proteins that provide a transmission of nerve impulse through the synapses. They are composed of a pentametric assembly of five homologous subunits (5 α7 subunits for α7nAChR, for example), oriented around the central pore. These receptors might be found in the chemical synapses of central and peripheral nervous system, and also in the neuromuscular synapses. Transmembrane domain of the one of such receptors constitutes ion channel. The conductive properties of ion channel strongly depend on the receptor conformation changes in the response of binding with some molecule, f.e. acetylcholine. Investigation of interaction between ligands and acetylcholine receptor is important for drug design. In this work we investigate theoretically the interaction between the set of different ligands (such as vanillin, thymoquinone, etc.) and the nicotinic acetylcholine receptor (primarily with subunit of the α7nAChR) by different methods and packages (AutodockVina, GROMACS, KVAZAR, HARLEM, VMD). We calculate interaction energy between different ligands in the subunit using molecular dynamics. On the base of obtained calculation results and using molecular docking we found an optimal location of different ligands in the subunit.

  7. Insensitive Acetylcholine Receptor Conferring Resistance of Plutella xylostella to Nereistoxin Insecticides

    CHENG Luo-gen; YU Guang; CHEN Zi-hao; LI Zhong-yin

    2008-01-01

    The combinative rate measurement of (3-[Ⅰ125] iodotyrosyl) α-bungarotoxin was applied in the analysis of the relation between nerve acetylcholine receptor and three types of insecticide resistance in diamondback moth, Plutella xylostella (L.). In the dimehypo-resistant strain and in the cartap-resistant strain, the nerve acetylcholine receptor showed the remarkable insensitivity to dimehypo and cartap, of which the binding rate to ligand was approximately 66 and 60%, respectively, of the susceptible strain. The sensitivity to deltamethrin in the deltamethrin-resistant strain did not show visible change. These results indicated that the decline in the sensitivity of nerve acetylcholine receptor to insecticide might be a potential mechanism to nereistoxin insecticides resistance in the diamondback moth.

  8. Functional Characterization of a Novel Class of Morantel-Sensitive Acetylcholine Receptors in Nematodes.

    Elise Courtot

    2015-12-01

    Full Text Available Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR.

  9. Inhibition of cortical acetylcholine release and cognitive performance by histamine H3 receptor activation in rats.

    Blandina, P.; Giorgetti, M.; L. Bartolini; M.Cecchi; Timmerman, H.; Leurs, R.; Pepeu, G; Giovannini, M. G.

    1996-01-01

    1. The effects of histamine and agents at histamine receptors on spontaneous and 100 mM K(+)-evoked release of acetylcholine, measured by microdialysis from the cortex of freely moving, rats, and on cognitive tests are described. 2. Local administration of histamine (0.1-100 microM) failed to affect spontaneous but inhibited 100 mM K(+)-stimulated release of acetylcholine up to about 50%. The H3 receptor agonists (R)-alpha-methylhistamine (RAMH) (0.1-10 microM), imetit (0.01-10 microM) and im...

  10. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-01

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies. PMID:26849939

  11. Blockage of muscle and neuronal nicotinic acetylcholine receptors by fluoxetine (Prozac)

    García-Colunga, J; Awad, J. N.; Miledi, R

    1997-01-01

    Fluoxetine (Prozac), a widely used antidepressant, is said to exert its medicinal effects almost exclusively by blocking the serotonin uptake systems. The present study shows that both muscle and neuronal nicotinic acetylcholine receptors are blocked, in a noncompetitive and voltage-dependent way, by fluoxetine, which also increases the rate of desensitization of the nicotinic receptors. Because these receptors are very widely distributed in the both central and peripheral nervous systems, th...

  12. Use of intact rat brain cells as a model to study regulation of muscarinic acetylcholine receptors

    Lee, J.H.; El-Fakahany, E.E.

    1985-08-12

    Intact rat brain cells were dissociated and used to study the regulation of muscarinic acetylcholine receptors upon exposure to muscarinic receptor agonists. Incubation of cells with carbamylcholine resulted in a time-dependent decrease in subsequent (/sup 3/H)N-methylscopolamine specific binding, an effect which reached a steady state after 3 hr at 37/sup 0/C. This effect of carbamylcholine was dependent on the concentration of the agonist in the incubation medium and was due to a reduction in the maximal binding capacity of the receptor with no decrease in the affinity of the remaining receptors. This preparation might be useful in future studies to elucidate the mechanisms underlying the regulation of muscarinic acetylcholine receptors in the central nervous system. 20 references, 3 tables.

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

    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

  14. Ca2+ is involved in muscarine-acetylcholine-receptor-mediated acetylcholine signal transduction in guard cells of Vicia faba L.

    MENG Fanxia; MIAO Long; ZHANG Shuqiu; LOU Chenghou

    2004-01-01

    Acetylcholine (ACh) is an important neurochemical transmitter in animals; it also exists in plants and plays a significant role in various kinds of physiological functions in plants. ACh has been known to induce the stomatal opening. By monitoring the changes of cytosolic Ca2+ with fluorescent probe Fluo-3 AM under the confocal microscopy,we found that exogenous ACh increased cytosolic Ca2+ concentration of guard cells of Vicia faba L. Muscarine, an agonist of muscarine acetylcholine receptor (mAChR), could do so as well. In contrast, atropine, the antagonist of mAChR abolished the ability of ACh to increase Ca2+ in guard cells.This mechanism is similar to mAChR in animals. When EGTA was used to chelate Ca2+ or ruthenium red to block Ca2+ released from vacuole respectively, the results showed that the increased cytosolic Ca2+ mainly come from intracellular Ca2+ store. The evidence supports that Ca2+ is involved in guard-cell response to ACh and that Ca2+ signal is coupled to mAChRs in ACh signal transduction in guard cells.

  15. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

    The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]Nicotine binds at equilibrium with Keq = 0.6 μM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the α- and γ-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the α-subunit was labeled via both agonist sites but the γ-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the α-subunit confirmed that Try-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193

  16. MUSCARINIC ACETYLCHOLINE RECEPTOR-EXPRESSION IN ASTROCYTES IN THE CORTEX OF YOUNG AND AGED RATS

    VANDERZEE, EA; DEJONG, GI; STROSBERG, AD; LUITEN, PGM

    1993-01-01

    The present report describes the cellular and subcellular distribution pattern of immunoreactivity to M35, a monoclonal antibody raised against purified muscarinic acetylcholine receptor protein, in astrocytes in the cerebral cortex of young and aged rats. Most M35-positive astrocytes were localized

  17. Synthesis and pharmacological evaluation of DHβE analogs as neuronal nicotinic acetylcholine receptor antagonists

    Jepsen, Tue H.; Jensen, Anders A.; Lund, Mads Henrik; Glibstrup, Emil; Kristensen, Jesper Langgaard

    2014-01-01

    Dihydro-β-erythroidine (DHβE) is a member of the Erythrina family of alkaloids and a potent competitive antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors (nAChRs). Guided by an X-ray structure of DHβE in complex with an ACh binding protein, we detail the design, synthesis, and...

  18. Tying up Nicotine: New Selective Competitive Antagonist of the Neuronal Nicotinic Acetylcholine Receptors

    Petersen, Ida Nymann; Crestey, François; Jensen, Anders A; Indurthi, Dinesh C; Pedersen, Henrik; Andreasen, Jesper T; Balle, Thomas; Kristensen, Jesper L

    2015-01-01

    Conformational restriction of the pyrrolidine nitrogen in nicotine by the introduction of an ethylene bridge provided a potent and selective antagonist of the α4β2-subtype of the nicotinic acetylcholine receptors. Resolution by chiral SFC, pharmacological characterization of the two enantiomers...

  19. Distinct neural pathways mediate alpha7 nicotinic acetylcholine receptor-dependent activation of the forebrain

    Thomsen, Morten S; Hay-Schmidt, Anders; Hansen, Henrik H; Mikkelsen, Jens D

    2010-01-01

    alpha(7) nicotinic acetylcholine receptor (nAChR) agonists are candidates for the treatment of cognitive deficits in schizophrenia. Selective alpha(7) nAChR agonists, such as SSR180711, activate neurons in the medial prefrontal cortex (mPFC) and nucleus accumbens shell (ACCshell) in rats, regions...

  20. alpha(7) Nicotinic acetylcholine receptor activation prevents behavioral and molecular changes induced by repeated phencyclidine treatment

    Thomsen, Morten Skøtt; Christensen, Ditte Z; Hansen, Henrik H; Redrobe, John P; Mikkelsen, Jens D

    determined in a modified Y-maze test. Polymorphisms in the alpha(7) nicotinic acetylcholine receptor (nAChR) gene have been linked to schizophrenia. Here we demonstrate that acute administration of the selective alpha(7) nAChR partial agonist SSR180711 dose-dependently reversed the behavioral impairment...

  1. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  2. Increases in muscle Ca2+ mediate changes in acetylcholinesterase and acetylcholine receptors caused by muscle contraction.

    Rubin, L L

    1985-01-01

    The synthesis of acetylcholinesterase (AcChoE; acetylcholine acetylhydrolase, EC 3.1.1.7) and of acetylcholine receptors (AcChoR) by cultured rat muscle fibers is influenced strongly by the level of muscle contractile activity. If fibers are grown in the presence of tetrodotoxin (TTX) to block spontaneous contraction, the total amount of AcChoE decreases markedly, as does the percentage of AcChoE assembled as the collagen-tailed presumed synaptic form of the enzyme. Under these conditions, ho...

  3. On homology modeling of the M-2 muscarinic acetylcholine receptor subtype

    Jakubík, Jan; Randáková, Alena; Doležal, Vladimír

    2013-01-01

    Roč. 27, č. 6 (2013), s. 525-538. ISSN 0920-654X R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptor * G-protein coupled receptor * homology energy estimation * MM-GBSA Subject RIV: ED - Physiology Impact factor: 2.782, year: 2013

  4. Nicotinic acetylcholine receptor: subunit structure, functional binding sites, and ion transport properties

    The structure of the nicotinic acetylcholine receptor has been highly conserved during animal evolution, and in all the species and tissues studied so far, including mammals, it is a pseudosymmetric, pentameric complex of related subunits with very similar physical properties. All subunits of these nicotinic receptors were derived from a common ancestral gene, probably by way of gene duplications occurring very early in animal evolution. 45 refs., 8 figs., 2 tabs

  5. CHRNB2 Is the Second Acetylcholine Receptor Subunit Associated with Autosomal Dominant Nocturnal Frontal Lobe Epilepsy*

    Phillips, Hilary A.; Favre, Isabelle; Kirkpatrick, Martin; Zuberi, Sameer M; Goudie, David; Heron, Sarah E.; Scheffer, Ingrid E.; Sutherland, Grant R.; Berkovic, Samuel F; Bertrand, Daniel; Mulley, John C

    2000-01-01

    Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is an uncommon, idiopathic partial epilepsy characterized by clusters of motor seizures occurring in sleep. We describe a mutation of the β2 subunit of the nicotinic acetylcholine receptor, effecting a V287M substitution within the M2 domain. The mutation, in an evolutionary conserved region of CHRNB2, is associated with ADNFLE in a Scottish family. Functional receptors with the V287M mutation are highly expressed in Xenopus oocytes ...

  6. CaMKIIα, a modulator of M4 muscarinic acetylcholine receptors

    Guo, Ming-Lei; Liu, Zhenguo; Chu, Xiang-Ping; Mao, Li-Min; WANG, John Q.

    2010-01-01

    G protein-coupled receptors (GPCRs) are subject to the regulation by protein kinases. By controlling the phosphorylation-dephosphorylation balance, protein kinases actively modify GPCR expression and function. In a recent study, we have identified a novel phosphorylation-dependent regulation of Gαi/o-coupled muscarinic acetylcholine receptors. A synapse-enriched protein kinase, Ca2+/calmodulin-dependent protein kinase II (CaMKIIα), binds directly and selectively to second intracellular loops ...

  7. Regional distribution of muscarinic acetylcholine receptors in the telencephalon of the pigeon (Columba livia f. domestica)

    The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using 3H-quinuclidinylbenzylate as a ligand. Highest receptor density was observed in the hyperstriatum ventrale, palaeostriatum augmentatum, septum, and parts of the archistriatum. In sites of known sensory input of neostriatum (field L) and ectostriatum low receptor binding was observed. Acetylcholinesterase distribution is in good agreement with the receptor picture only in the basal telencephalon. In the pallium differences in the pattern of these two components can be seen. (author)

  8. Expression of somatostatin receptor genes and acetylcholine receptor development in rat skeletal muscle during postnatal development.

    Peng, M; Conforti, L; Millhorn, D E

    1998-05-01

    Our laboratory reported previously that somatostatin (SST) is transiently expressed in rat motoneurons during the first 14 days after birth. We investigated the possibility that the SST receptor (SSTR) is expressed in skeletal muscle. We found that two of the five subtypes of SSTR (SSTR3 and SSTR4) are expressed in skeletal muscle with a time course that correlates with the transient expression of SST in motoneurons. In addition, SSTR2A is expressed from birth to adulthood in skeletal muscle. Both SSTR2A and SSTR4 are also expressed in L6 cells, a skeletal muscle cell line. Somatostatin acting through its receptors has been shown to stimulate tyrosine phosphatase activity in a number of different tissues. We found that several proteins (50, 65, 90, 140, 180 and 200 kDa) exhibited a reduced degree of tyrosine phosphorylation following SST treatment. Inhibition of tyrosine phosphatase activity with sodium orthovanadate increased expression of the nicotinic acetyl-choline receptor (nAChR) epsilon subunit mRNA by three fold. Somatostatin reversed the elevated epsilon mRNA following orthovanadate treatment. These findings show that SSTR is expressed in skeletal muscle and that SST acting via the SSTR regulates tyrosine phosphorylation and expression of the epsilon subunit of the AChR in the rat skeletal muscle. PMID:9852305

  9. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor α subunit

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the α subunit of the receptor, with little or no change in the levels of γ- and δ-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of α-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of α subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of α-subunit mRNA, with little change in the amount of γ- and δ-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the α-subunit nuclear precursor

  10. Selective effects of carbamate pesticides on rat neuronal nicotinic acetylcholine receptors and rat brain acetylcholinesterase

    Effects of commonly used carbamate pesticides on rat neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes have been investigated using the two-electrode voltage clamp technique. The potencies of these effects have been compared to the potencies of the carbamates to inhibit rat brain acetylcholinesterase. The potency order of six carbamates to inhibit α4β4 nicotinic receptors is fenoxycarb > EPTC > carbaryl, bendiocarb > propoxur > aldicarb with IC50 values ranging from 3 μM for fenoxycarb to 165 μM for propoxur and >1 mM for aldicarb. Conversely, the potency order of these carbamates to inhibit rat brain acetylcholinesterase is bendiocarb > propoxur, aldicarb > carbaryl >> EPTC, fenoxycarb with IC50 values ranging from 1 μM for bendiocarb to 17 μM for carbaryl and >>1 mM for EPTC and fenoxycarb. The α4β2, α3β4, and α3β2 nicotinic acetylcholine receptors are inhibited by fenoxycarb, EPTC, and carbaryl with potency orders similar to that for α4β4 receptors. Comparing the potencies of inhibition of the distinct subtypes of nicotinic acetylcholine receptors shows that the α3β2 receptor is less sensitive to inhibition by fenoxycarb and EPTC. The potency of inhibition depends on the carbamate as well as on a combination of α and β subunit properties. It is concluded that carbamate pesticides affect different subtypes of neuronal nicotinic receptors independently of acetylcholinesterase inhibition. This implicates that neuronal nicotinic receptors are additional targets for some carbamate pesticides and that these receptors may contribute to carbamate pesticide toxicology, especially after long-term exposure

  11. Evidence for the extramembranous location of the putative amphipathic helix of acetylcholine receptor

    Evidence has been obtained demonstrating that the peptides GVKYIAE and AIKYIAE found in the potential amphipathic helices of the α and β subunits, respectively, of acetylcholine receptor are not buried in the membrane. The peptide KYIAE was synthesized, and polyclonal antibodies were prepared against a conjugate of bovine serum albumin and synthetic peptide. An immunoadsorbent capable of binding and subsequently releasing peptides ending with the sequence-YIAE was produced by attaching these specific antibodies to agarose. Native acetylcholine receptor was labeled with pyridoxal phosphate and Na[3H]BH4. The labeled protein was stripped of phospholipid and digested with the protease from Staphylococcus aureus strain V8. The digest was submitted to immunoadsorption to isolate the labeled indigenous peptides. As a control, α and β polypeptides prepared by gel filtration of a solution of acetylcholine receptor in detergent were stripped of detergent and labeled with pyridoxal phosphate and Na[3H]BH4 in the presence of 8 M urea. The labeled α and β polypeptides were digested and submitted to immunoadsorption. The specific radioactivities of the indigenous peptides from the α and β subunits labeled under native and denaturing conditions were nearly equal. In similar experiments using isethionyl (2',4'-dinitrophenyl)-3-aminopropionimidate as the labeling agent, the indigenous peptides from native and denatured receptor were also labeled to the same extent. Since these peptides are labeled to the same extent whether or not the protein is denatured, they cannot be buried in the membrane

  12. Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

    Thal, David M; Sun, Bingfa; Feng, Dan; Nawaratne, Vindhya; Leach, Katie; Felder, Christian C; Bures, Mark G; Evans, David A; Weis, William I; Bachhawat, Priti; Kobilka, Tong Sun; Sexton, Patrick M; Kobilka, Brian K; Christopoulos, Arthur

    2016-03-17

    Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains. PMID:26958838

  13. Anti-acetylcholine receptor antibody titres in the sera of myasthenia patients treated with plasma exchange combined with immunosuppressive therapy.

    Carter, B.; Harrison, R.; Lunt, G G; Behan, P O; Simpson, J. A.

    1980-01-01

    Anti-acetylcholine receptor antibody titres have been monitored in the sera of 19 myasthenic patients treated with plasma exchange combined with a three month period of immunosuppressive therapy. In general the post-exchange titres stabilised at below pre-exchange levels for prolonged periods which were associated with clinical improvement. In seven instances recurrence of symptoms occurred and in six of these cases relapse was shown to be associated with a rise in anti-acetylcholine receptor...

  14. A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.

    Maelle Jospin

    2009-12-01

    Full Text Available In the nematode Caenorhabditis elegans, cholinergic motor neurons stimulate muscle contraction as well as activate GABAergic motor neurons that inhibit contraction of the contralateral muscles. Here, we describe the composition of an ionotropic acetylcholine receptor that is required to maintain excitation of the cholinergic motor neurons. We identified a gain-of-function mutation that leads to spontaneous muscle convulsions. The mutation is in the pore domain of the ACR-2 acetylcholine receptor subunit and is identical to a hyperactivating mutation in the muscle receptor of patients with myasthenia gravis. Screens for suppressors of the convulsion phenotype led to the identification of other receptor subunits. Cell-specific rescue experiments indicate that these subunits function in the cholinergic motor neurons. Expression of these subunits in Xenopus oocytes demonstrates that the functional receptor is comprised of three alpha-subunits, UNC-38, UNC-63 and ACR-12, and two non-alpha-subunits, ACR-2 and ACR-3. Although this receptor exhibits a partially overlapping subunit composition with the C. elegans muscle acetylcholine receptor, it shows distinct pharmacology. Recordings from intact animals demonstrate that loss-of-function mutations in acr-2 reduce the excitability of the cholinergic motor neurons. By contrast, the acr-2(gf mutation leads to a hyperactivation of cholinergic motor neurons and an inactivation of downstream GABAergic motor neurons in a calcium dependent manner. Presumably, this imbalance between excitatory and inhibitory input into muscles leads to convulsions. These data indicate that the ACR-2 receptor is important for the coordinated excitation and inhibition of body muscles underlying sinusoidal movement.

  15. Expression of the α7 nicotinic acetylcholine receptor in human lung cells

    Schuller Hildegard M

    2005-04-01

    Full Text Available Abstract Background We and others have shown that one of the mechanisms of growth regulation of small cell lung cancer cell lines and cultured pulmonary neuroendocrine cells is by the binding of agonists to the α7 neuronal nicotinic acetylcholine receptor. In addition, we have shown that the nicotine-derived carcinogenic nitrosamine, 4(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK, is a high affinity agonist for the α7 nicotinic acetylcholine receptor. In the present study, our goal was to determine the extent of α7 mRNA and protein expression in the human lung. Methods Experiments were done using reverse transcription polymerase chain reaction (RT-PCR, a nuclease protection assay and western blotting using membrane proteins. Results We detected mRNA for the neuronal nicotinic acetylcholine receptor α7 receptor in seven small cell lung cancer (SCLC cell lines, in two pulmonary adenocarcinoma cell lines, in cultured normal human small airway epithelial cells (SAEC, one carcinoid cell line, three squamous cell lines and tissue samples from nine patients with various types of lung cancer. A nuclease protection assay showed prominent levels of α7 in the NCI-H82 SCLC cell line while α7 was not detected in SAEC, suggesting that α7 mRNA levels may be higher in SCLC compared to normal cells. Using a specific antibody to the α7 nicotinic receptor, protein expression of α7 was determined. All SCLC cell lines except NCI-H187 expressed protein for the α7 receptor. In the non-SCLC cells and normal cells that express the α7 nAChR mRNA, only in SAEC, A549 and NCI-H226 was expression of the α7 nicotinic receptor protein shown. When NCI-H69 SCLC cell line was exposed to 100 pm NNK, protein expression of the α7 receptor was increased at 60 and 150 min. Conclusion Expression of mRNA for the neuronal nicotinic acetylcholine receptor α7 seems to be ubiquitously expressed in all human lung cancer cell lines tested (except for NCI-H441 as well as normal

  16. Regulation of Synaptic Transmission and Plasticity by Neuronal Nicotinic Acetylcholine Receptors

    McKay, Bruce E.; Placzek, Andon N; Dani, John A.

    2007-01-01

    Nicotinic acetylcholine receptors (nAChRs) are widely expressed throughout the central nervous system and participate in a variety of physiological functions. Recent advances have revealed roles of nAChRs in the regulation of synaptic transmission and synaptic plasticity, particularly in the hippocampus and midbrain dopamine centers. In general, activation of nAChRs causes membrane depolarization and directly and indirectly increases the intracellular calcium concentration. Thus, when nAChRs ...

  17. M2 Muscarinic acetylcholine receptor modulates rat airway smooth muscle cell proliferation

    Placeres-Uray, Fabiola A; Febres-Aldana, Christopher A; Fernandez-Ruiz, Ruth; Gonzalez de Alfonzo, Ramona; Lippo de Becemberg, Itala A; Alfonzo, Marcelo J

    2013-01-01

    Airways chronic inflammatory conditions in asthma and COPD are characterized by tissue remodeling, being smooth muscle hyperplasia, the most important feature. Non-neuronal and neuronal Acetylcholine acting on muscarinic receptors (MAChRs) has been postulated as determinant of tissue remodeling in asthma and COPD by promoting proliferation and phenotypic changes of airway smooth muscle cells (ASMC). The objective was to evaluate proliferative responses to muscarinic agonist as carbamylcholine...

  18. Neonicotinoid Binding, Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis; Thany, Steeve H.; Tricoire-Leignel, Helene

    2014-01-01

    Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and ...

  19. Minor structural changes in nicotinoid insecticides confer differential subtype selectivity for mammalian nicotinic acetylcholine receptors

    Tomizawa, Motohiro; Casida, John E.

    1999-01-01

    The major nitroimine insecticide imidacloprid (IMI) and the nicotinic analgesics epibatidine and ABT-594 contain the 6-chloro-3-pyridinyl moiety important for high activity and/or selectivity. ABT-594 has considerable nicotinic acetylcholine receptor (AChR) subtype specificity which might carry over to the chloropyridinyl insecticides. This study considers nine IMI analogues for selectivity in binding to immuno-isolated α1, α3 and α7 containing nicotinic AChRs and to purported α4β2 nicotinic ...

  20. Utilization of Superfused Cerebral Slices in Probing Muscarinic Receptor Autoregulation of Acetylcholine Release

    Alquicer, Glenda; Doležal, Vladimír; El-Fakahany, E. E.

    New York: Springer, 2016 - (Mysliveček, J.; Jakubík, J.), s. 221-233. (Neuromethods. 107). ISBN 978-1-4939-2857-6 R&D Projects: GA ČR(CZ) GA14-05696S; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic receptors * acetylcholine release * autoregulation * superfusion Subject RIV: FH - Neurology

  1. Variants in nicotinic acetylcholine receptors α5 and α3 increase risks to nicotine dependence†

    Chen, Xiangning; Chen, Jingchun; Williamson, Vernell S; An, Seon-Sook; Hettema, John M.; Aggen, Steven H.; Neale, Michael C.; Kendler, Kenneth S.

    2009-01-01

    Nicotinic acetylcholine receptors bind to nicotine and initiate the physiological and pharmacological responses to tobacco smoking. In this report, we studied the association of α5 and α3 subunits with nicotine dependence and with the symptoms of alcohol and cannabis abuse and dependence in two independent epidemiological samples (n = 815 and 1,121, respectively). In this study, seven single nucleotide polymorphisms were genotyped in the CHRNA5 and CHRNA3 genes. In both samples, we found that...

  2. Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms

    Randáková, Alena; Dolejší, Eva; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; El-Fakahany, E. E.; Jakubík, Jan

    2015-01-01

    Roč. 97, Jul 2015 (2015), s. 27-39. ISSN 1043-6618 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) EE2.3.30.0025 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * atypical agonists * xanomeline * activation mechanism Subject RIV: ED - Physiology Impact factor: 4.408, year: 2014

  3. Isolation of acetylcholine receptor clusters in substrate-associated material from cultured rat myotubes using saponin

    1984-01-01

    After exposure of rat myotube cultures to saponin, less than 1% of the cellular protein was found to remain associated with the tissue culture substrate. This substrate-associated material contained approximately 10% of the acetylcholine receptors (AChRs) and greater than 80% of the large, ventral AChR clusters present in the original culture. The domain structure evident in intact cells was maintained in AChR clusters after isolation using saponin. However, vinculin, present at the clusters ...

  4. Quantitative Molecular Imaging of Neuronal Nicotinic Acetylcholine Receptors in the Human Brain with A-85380 Radiotracers

    Lotfipour, Shahrdad; Mandelkern, Mark; Brody, Arthur L.

    2011-01-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) have been implicated in a spectrum of cognitive functions as well as psychiatric and neurodegenerative disorders, including tobacco addiction and Alzheimer's Disease. The examination of neuronal nAChRs in living humans is a relatively new field. Researchers have developed brain-imaging radiotracers for nAChRs, with radiolabeled A-85380 compounds having the most widespread use. We provide a brief background on nAChRs, followed by a discussion...

  5. Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes

    1984-01-01

    Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX...

  6. Muscarinic acetylcholine receptor subtypes: localization and structure/function

    Brann, M R; Ellis, J; Jørgensen, H;

    1993-01-01

    Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The...... are described, as well as the implied structures of these functional domains....

  7. Characterization of a putative acetylcholine receptor in chick ciliary ganglion neurons

    Monoclonal antibodies to the main immunogenic region on the alpha subunit of acetylcholine receptors in muscle and electric organ recognize membrane components in chick brain and ciliary ganglia that are candidates for the neuronal receptor. The component in chick brain has been purified by immunoaffinity chromatography. It specifically binds nicotine but not alpha-bungarotoxin, and can be affinity labeled with (3H)bromoacetylcholine. The cross-reacting component in ciliary ganglion neurons is concentrated in synaptic membrane, and can be modulated by exposure of the cells to cholinergic ligands in culture. The cross-reacting component in ciliary ganglion neurons is an integral membrane component that binds concanavalin A, and it is distinct from the alpha-bungarotoxin binding component. The acetylcholine receptor function in these neurons can be locked by affinity alkylation with bromoacetylcholine, indicating similarity in this respect to receptors from muscle and electric organ. Antisera raised against the partially purified component from chick brain also block receptor function on ciliary ganglion neurons. The subcellular distribution of the ganglion component in culture is assessed, and it is shown that approximately 2/3 of the cross-reacting components are intracellular; the majority of these seem not to be destined for insertion into the plasma membrane

  8. Changes in acetylcholine content, release and muscarinic receptors in rat hippocampus under cold stress

    The aim was to study the mechanism of the previously established decrease in acetylcholine (ACh) concentration in the rat hippocampus under cold stress. Male rats were exposed for 14 days to cold (5 degree C) or kept (controls) at room temperature (24 degree C). Acetylcholine content, release and muscarinic receptor binding were investigated in the hippocampus. Cold exposure resulted in a decrease of ACh concentration in the dorsal hippocampus. Moreover, the potassium-evoked release of ACh from hippocampal slices was increased and an increase of maximal binding capacity of [3H](-) quinuclidinyl benzilate in the dorsal hippocampus of cold exposed animals was also observed. Thus the decrease of hippocampal ACh concentration under cold exposure is probably due to its increased release. On balance then, our results demonstrate that cold stress in the rat induces significant activation of the hippocampal cholinergic system

  9. Positive allosteric action of eburnamonine on cardiac muscarinic acetylcholine receptors.

    Proska, J; Tucek, S

    1996-06-01

    It was discovered recently that alcuronium and strychnine (which is a precursor of alcuronium) allosterically increase the affinity of cardiac muscarinic receptors for the antagonist, N-methylscopolamine. We have now investigated the effects of l-eburnamonine and vincamine, which are both closely related to strychnine. In experiments on rat heart atria, l-eburnamonine was found to increase the binding of [3H]N-methylscopolamine with Ehlert's cooperativity coefficient alpha = 0.35, which indicates that the strength of its allosteric action is close to that of alcuronium and strychnine (alpha = 0.31 and 0.44, respectively). However, the affinity of l-eburnamonine for the cardiac muscarinic receptors is lower than the affinities of alcuronium and strychnine (KAR = 22.6 microM, 0.15 microM, and 3.4 microM, respectively). In spite of its extremely close similarity to l-eburnamonine, vincamine has a negative allosteric effect on the binding of [3H]N-methylscopolamine (alpha = 4.1; KAR = 22.8 microM). It is likely that a systematic investigation of the allosteric effects of the analogues of strychnine will not only yield new allosteric effectors on muscarinic receptors, but also clarify the structural features responsible for the direction (positive or negative) of their allosteric effect. PMID:8813554

  10. Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

    Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Gαq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Gαi/o-protein coupled M2 receptor activity mediated neuronal differentiation

  11. Effects of alpha-7 nicotinic acetylcholine receptor positive allosteric modulator on lipopolysaccharide-induced neuroinflammatory pain in mice.

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

    Evidence indicates that microglial activation contributes to the pathophysiology and maintenance of neuroinflammatory pain involving central nervous system alpha-7 nicotinic acetylcholine receptors. The objective of the present study was to determine the effects of 3a,4,5,9b-Tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide (TQS), an alpha-7 nicotinic acetylcholine receptor positive allosteric modulator (PAM), on tactile allodynia and thermal hyperalgesia following lipopolysaccharide (LPS)-induced microglial activation in hippocampus, a neuroinflammatory pain model in mice. In addition, we examined the effects of TQS on microglial activation marker, an ionized calcium-binding adapter molecule 1 (Iba-1), in the hippocampus may be associated with neuroinflammatory pain. Pretreatment of TQS (4mg/kg) significantly reduced LPS (1mg/kg)-induced tactile allodynia and thermal hyperalgesia. Moreover, pretreatment of methyllycaconitine (3mg/kg) significantly reversed TQS-induced antiallodynic and antihyperalgesic responses indicating the involvement of alpha-7 nicotinic acetylcholine receptor. Pretreatment of TQS significantly decreased LPS-induced increased in hippocampal Iba-1 expression. Overall, these results suggest that TQS reduces LPS-induced neuroinflammatory pain like symptoms via modulating microglial activation likely in the hippocampus and/or other brain region by targeting alpha-7 nicotinic acetylcholine receptor. Therefore, alpha-7 nicotinic acetylcholine receptor PAM such as TQS could be a potential drug candidate for the treatment of neuroinflammatory pain. PMID:27154173

  12. An allosteric enhancer of M4muscarinic acetylcholine receptor function inhibits behavioral and neurochemical effects of cocaine

    Dencker, Ditte; Weikop, Pia; Sørensen, Gunnar;

    2012-01-01

    The mesostriatal dopamine system plays a key role in mediating the reinforcing effects of psychostimulant drugs like cocaine. The muscarinic M4 acetylcholine receptor subtype is centrally involved in the regulation of dopamine release in striatal areas. Consequently, striatal M4 receptors could be...

  13. A subpopulation of neuronal M4 muscarinic acetylcholine receptors plays a critical role in modulating dopamine-dependent behaviors

    Jeon, Jongrye; Nielsen, Ditte Dencker; Wörtwein, Gitta;

    2010-01-01

    Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M(1)-M(5) mAChRs). Like other mAChR subtypes, the M(4) mAChR is widely expressed in different regions of the forebrain. Interestingly, M(4) mAChRs are coexpres...

  14. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists. PMID:27055770

  15. Activation of muscarinic acetylcholine receptors elicits pigment granule dispersion in retinal pigment epithelium isolated from bluegill

    Crittenden Elizabeth L

    2004-07-01

    Full Text Available Abstract Background In fish, melanin pigment granules in the retinal pigment epithelium disperse into apical projections as part of the suite of responses the eye makes to bright light conditions. This pigment granule dispersion serves to reduce photobleaching and occurs in response to neurochemicals secreted by the retina. Previous work has shown that acetylcholine may be involved in inducing light-adaptive pigment dispersion. Acetylcholine receptors are of two main types, nicotinic and muscarinic. Muscarinic receptors are in the G-protein coupled receptor superfamily, and five different muscarinic receptors have been molecularly cloned in human. These receptors are coupled to adenylyl cyclase, calcium mobilization and ion channel activation. To determine the receptor pathway involved in eliciting pigment granule migration, we isolated retinal pigment epithelium from bluegill and subjected it to a battery of cholinergic agents. Results The general cholinergic agonist carbachol induces pigment granule dispersion in isolated retinal pigment epithelium. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M1 antagonist pirenzepine, and by the M3 antagonist 4-DAMP. Pigment granule dispersion was also induced by the M1 agonist 4-[N-(4-chlorophenyl carbamoyloxy]-4-pent-2-ammonium iodide. In contrast the M2 antagonist AF-DX 116 and the M4 antagonist tropicamide failed to block carbachol-induced dispersion, and the M2 agonist arecaidine but-2-ynyl ester tosylate failed to elicit dispersion. Conclusions Our results suggest that carbachol-mediated pigment granule dispersion occurs through the activation of Modd muscarinic receptors, which in other systems couple to phosphoinositide hydrolysis and elevation of intracellular calcium. This conclusion must be corroborated by molecular studies, but suggests Ca2+-dependent pathways may be involved in light-adaptive pigment dispersion.

  16. Stimulation of brain muscarinic acetylcholine receptors acutely reverses radiogenic hypodipsia

    A sufficiently large dose of ionizing radiation produces changes in water consumption. However, the direction, durations, and physiological substrates of these alterations remain in question. Here we report a 5-d hypodipsia in rats exposed to 600 rads 60Co but a more transient, albeit larger, reduction in drinking after 1000 60Co. Brain cholinergic neurons have been implicated as mediators of thirst. Therefore, we explored the role of hypothalamic muscarinic receptors in the production of radiation-induced hypodipsia. This was accomplished through the intrahypothalamic injection of carbachol (a muscarinic agonist) or atropine (a muscarinic antagonist) in irradiated rats. Intracranial carbachol produced acute reversal of radiogenic hypodipsia while atropine potentiated the hypodipsia. These post-irradiation drug-induced behaviors were similar to those observed after the same drug treatments before irradiation. Since cholinergic neuronal functions persist and are labile (can be pharmacologically stimulated and blocked) after irradiation, this suggests that other neuronal systems and/or neurochemicals may be more prominently involved in radiogenic hypodipsia

  17. Regulation of phosphorylation of nicotinic acetylcholine receptors in mouse BC3H1 myocytes

    Smith, M.M.; Merlie, J.P.; Lawrence, J.C. Jr.

    1987-09-01

    By using /sup 32/P-labeling methods and performing immunoprecipitations with specific antibodies, the authors have found that three subunits of the nicotinic acetylcholine receptor and phosphorylated in mouse skeletal muscle cells. In nonstimulated cells, the molar ratios of phosphate estimated in ..cap alpha.., ..beta.., and delta subunits were 0.02, 0.05, and 0.5, respectively. All three subunits contained predominantly phosphoserine with some phosphothreonine; the ..beta.., subunit also contained phosphotyrosine. Incubating cells with agents that stimulate cAMP-dependent pathways (isoproterenol, forskolin, 8-Br-cAMP) increased the phosphorylation of the delta subunit by 50%, but phosphate labeling of the ..beta.. subunit was depressed by a third. In contrast, when cells were incubated with the divalent cation ionophores A-23187 or ionomycin, phosphorylation of both the delta and ..beta.. subunits increased. The results indicate that acetylcholine receptors are phosphorylated to significant levels in skeletal muscle cells and that cAMP-dependent and Ca/sup 2 +/-dependent pathways exist for controlling the phosphorylation state of the receptor subunits.

  18. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori

    Zhang Chuan-Xi; Dong Ke; Shao Ya-Ming

    2007-01-01

    Abstract Background Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based o...

  19. Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Feuerbach, Dominik; Targowska-Duda, Katarzyna M.; Maciejewski, Ryszard; Jozwiak, Krzysztof; Moaddel, Ruin; Glick, Stanley D.; Wainer, Irving W.

    2010-01-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (±)-epibatidine-induced AChR Ca2+ influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [3H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in...

  20. Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.

    2010-01-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca2+ influx in hα3β4 AChRs with ~9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single s...

  1. Block by acetylcholine of mouse muscle nicotinic receptors, stably expressed in fibroblasts

    1995-01-01

    We have measured the concentration and voltage dependence of block by acetylcholine (ACh) of fetal- and adult-type mouse muscle nicotinic receptors, expressed in a fibroblast cell line. Data, obtained at a transmembrane potential of -60 mV and with ACh concentrations of 1 mM and above, are broadly consistent with the occlusion of an open channel with a single ACh+ ion (simple open channel block). The rate of recovery from block is approximately 40,000s-1 and has only a weak voltage dependence...

  2. m1 Acetylcholine Receptor Expression is Decreased in Hippocampal CA1 region of Aged Epileptic Animals

    Cavarsan, Clarissa Fantin; Avanzi, Renata Della Torre; Queiroz, Claudio Marcos; Xavier, Gilberto Fernando; Mello, Luiz Eugênio; Covolan, Luciene

    2011-01-01

    In the present study, we investigated the possible additive effects of epilepsy and aging on the expression of m1 muscarinic acetylcholine receptors (AChR) in the rat hippocampus. Young (3 months) and Aged (20 months) male, Wistar rats were treated with pilocarpine to induce status epilepticus (SE). Immunohistochemical procedure for m1 AChR detection was performed 2 months after pilocarpine-induced SE. In the CA1 pyramidal region m1 AChR staining was significantly decreased in aged epileptic ...

  3. Prostate stem cell antigen interacts with nicotinic acetylcholine receptors and is affected in Alzheimer's disease

    Jensen, Majbrit Myrup; Mikkelsen, Jens D.; Arvaniti, Maria; Pinborg, Lars Hageman; Thomsen, Morten Skøtt

    2015-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder involving impaired cholinergic neurotransmission and dysregulation of nicotinic acetylcholine receptors (nAChRs). Ly-6/neurotoxin (Lynx) proteins have been shown to modulate cognition and neural plasticity by binding to nAChR subtypes and...... are present in the human brain. We further showed that PSCA forms stable complexes with the α4 nAChR subunit and decreases nicotine-induced extracellular-signal regulated kinase phosphorylation in PC12 cells. In addition, we analyzed protein levels of PSCA and Lypd6 in postmortem tissue of medial...

  4. alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief

    McGranahan, Tresa M.; Patzlaff, Natalie E.; Grady, Sharon R; Heinemann, Stephen F.; Booker, T.K.

    2011-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbra...

  5. The role of alpha4 containing nicotinic acetylcholine receptors in dopamine neurons

    McGranahan, Tresa Michelle

    2011-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and, thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4- containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midb...

  6. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Pharmacological Properties of 3’- (Substituted phenyl) Deschloroepibatidine Analogs

    F. Ivy Carroll; Yokota, Yasuno; Ma, Wei; Lee, Jeffrey R.; Brieaddy, Lawrence E.; Burgess, Jason P.; Navarro, Hernán A.; Damaj, M. I.; Martin, Billy R.

    2007-01-01

    A series of 3’-(substituted phenyl)deschloroepibatidine analogs (5a–j) were synthesized. The α4β2* and α7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a–j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13) the partial agonist, varenicline (3) and the antagonist 2’-fluoro-3’-(substituted phenyl)deschloroepibatidine analogs...

  7. Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid, Acyrthosiphon pisum

    LIU Yi-peng; LIN Ke-jian; LIU Yang; GUI Fu-rong; WANG Gui-rong

    2013-01-01

    The nicotinic acetylcholine receptors (nAchRs) are cholinergic receptors that form ligand-gated ion channels by ifve subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identiifed and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression proifles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.

  8. Topological dispositions of lysine α380 and lysine γ486 in the acetylcholine receptor from Torpedo californica

    The locations have been determined, with respect to the plasma membrane, of lysine α380 and lysine γ486 in the α subunit and the γ subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the α subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the γ subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium [3H]-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine α380 and lysine γ486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine α380 is on the inside surface of a vesicle and lysine γ486 is on the outside surface. Because a majority (85%) of the total binding sites for α-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine α380 and lysine γ486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor

  9. Topological dispositions of lysine. alpha. 380 and lysine. gamma. 486 in the acetylcholine receptor from Torpedo californica

    Dwyer, B.P. (Univ. of California, San Diego, La Jolla (USA))

    1991-04-23

    The locations have been determined, with respect to the plasma membrane, of lysine {alpha}380 and lysine {gamma}486 in the {alpha} subunit and the {gamma} subunit, respectively, of the nicotinic acetylcholine receptor from Torpedo californica. Immunoadsorbents were constructed that recognize the carboxy terminus of the peptide GVKYIAE released by proteolytic digestion from positions 378-384 in the amino acid sequence of the {alpha} subunit of the acetylcholine receptor and the carboxy terminus of the peptide KYVP released by proteolytic digestion from positions 486-489 in the amino acid sequence of the {gamma} subunit. They were used to isolate these peptides from proteolytic digests of polypeptides from the acetylcholine receptor. Sealed vesicles containing the native acetylcholine receptor were labeled with pyridoxal phosphate and sodium ({sup 3}H)-borohydride. The effect of saponin on the incorporation of pyridoxamine phosphate into lysine {alpha}380 and lysine {gamma}486 from the acetylcholine receptor in these vesicles was assessed with the immunoadsorbents. The conclusions that follow from these results are that lysine {alpha}380 is on the inside surface of a vesicle and lysine {gamma}486 is on the outside surface. Because a majority (85%) of the total binding sites for {alpha}-bungarotoxin bind the toxin in the absence of saponin, the majority of the vesicles are right side out with the inside of the vesicle corresponding to the cytoplasmic surface and the outside of the vesicle corresponding to the extracytoplasmic, synaptic surface. Because lysine {alpha}380 and lysine {gamma}486 lie on opposite sides of the membrane, a membrane-spanning segment must be located between the two positions occupied by these two amino acids in the common sequence of a polypeptide of the acetylcholine receptor.

  10. Contrasting Effects of Allosteric and Orthosteric Agonists on M1 Muscarinic Acetylcholine Receptor Internalization and Down-regulation

    Thomas, Rachel L.; Christopher J Langmead; Wood, Martyn D; Challiss, R.A. John

    2009-01-01

    A new class of subtype-selective muscarinic acetylcholine (mACh) receptor agonist that activates the receptor through interaction at a site distinct from the orthosteric acetylcholine binding site has been reported recently. Here, we have compared the effects of orthosteric (oxotremorine-M, arecoline, pilocarpine) and allosteric [4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine (AC-42); 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1)] agonists on M1 mAC...

  11. Nicotinic acetylcholine receptor polymorphism, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases: a cohort study

    Kaur-Knudsen, Diljit; Bojesen, Stig E; Tybjærg-Hansen, Anne; Nordestgaard, Børge G

    2011-01-01

    We examined the associations between the nicotinic acetylcholine receptor polymorphism (rs1051730) on chromosome 15q25 marking the gene cluster CHRNA3-CHRNB4-CHRNA5, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases in the general population.......We examined the associations between the nicotinic acetylcholine receptor polymorphism (rs1051730) on chromosome 15q25 marking the gene cluster CHRNA3-CHRNB4-CHRNA5, smoking behavior, and tobacco-related cancer and lung and cardiovascular diseases in the general population....

  12. Aspects of dopamine and acetylcholine release induced by glutamate receptors; Aspectos das liberacoes de dopamina e acetilcolina mediadas por receptores de glutamato

    Paes, Paulo Cesar de Arruda

    2002-07-01

    The basal ganglia play an important role in the motor control of rats and humans. This control involves different neurotransmitters and the mutual control of these key elements has been subject to several studies. In this work we determined the role of glutamate on the release of radioactively labelled dopamine and acetylcholine from chopped striatal tissue in vitro. The values of Effective Concentration 50% for glutamate, NMDA, kainic, quisqualic acids and AMPA on the release of dopamine and acetylcholine were obtained. The inhibitory effects of magnesium, tetrodotoxin, MK-801, AP5 and MCPG, as well as the effects of glycin were evaluated. The results suggested that dopamine is influenced by the NMDA type glutamate receptor while acetylcholine seems to be influenced by NMDA, kainate and AMPA receptors. Tetrodotoxin experiments suggested that kainate receptors are both present in cholinergic terminals and cell bodies while AMPA and NMDA receptors are preferentially distributed in cell bodies. Magnesium effectively blocked the NMDA stimulation and unexpectedly also AMPA- and quisqualate-induced acetylcholine release. The latter could not be blocked by MCPG ruling out the participation of methabotropic receptors. MK-801 also blocked NMDA-receptors. Results point out the importance of the glutamic acid control of dopamine and acetylcholine release in striatal tissue. (author)

  13. The Anti-Acetylcholine Receptor Antibody Test in Suspected Ocular Myasthenia Gravis

    Jung Jin Lee

    2014-01-01

    Full Text Available Aim. To estimate the clinical significance of anti-acetylcholine receptor antibody (anti-AChR-Ab levels in suspected ocular myasthenia gravis. Methods. In total, 144 patients complaining of fluctuating diplopia and ptosis were evaluated for serum levels of anti-acetylcholine receptor antibody and their medical charts were retrospectively reviewed. Subjects were classified into three groups: variable diplopia only, ptosis only, and both variable diplopia and ptosis. We investigated serum anti-AChR-Ab titer levels and performed thyroid autoantibody tests. Results. Patients’ chief complaints were diplopia (N=103, ptosis (N=12, and their concurrence (N=29. Abnormal anti-AChR-Ab was observed in 21 of 144 patients (14.1%. Between the three groups, mean age, number of seropositive patients, and mean anti-AChR-Ab level were not significantly different (P=0.224, 0.073, and 0.062, resp.. Overall, 27.5% of patients had abnormal thyroid autoantibodies. Conclusion. The sensitivity of anti-AChR-Ab was 14.1% in suspected ocular myasthenia gravis and seropositivity in myasthenia gravis patients showed a high correlation with the presence of thyroid autoantibodies.

  14. Muscarinic acetylcholine receptor down-regulation limits the extent of inhibition of cell cycle progression in Chinese hamster ovary cells.

    Detjen, K.; Yang, J; Logsdon, C D

    1995-01-01

    Cellular desensitization is believed to be important for growth control but direct evidence is lacking. In the current study we compared effects of wild-type and down-regulation-resistant mutant m3 muscarinic receptors on Chinese hamster ovary (CHO-K1) cell desensitization, proliferation, and transformation. We found that down-regulation of m3 muscarinic acetylcholine receptors was the principal mechanism of desensitization of receptor-activated inositol phosphate phospholipid hydrolysis in t...

  15. The M4 muscarinic acetylcholine receptor play a key role in the control of murine hair follicle cycling and pigmentation

    Hasse, Sybille; Chernyavsky, Alex I; Grando, Sergei A.; Paus, Ralf

    2007-01-01

    Cholinergic receptors of the muscarinic class (M1-M5) are expressed in epidermal keratinocytes and melanocytes as well as in the hair follicle. Knockout (KO) mice of all five receptors have been created and resulted in different phenotypes. KO mice with a deletion of the M4 muscarinic acetylcholine receptor (M4R) present a striking hair phenotype, which we have analyzed here in greater detail by quantitative histomorphometry. Earlier studies revealed a retarded hair follicle morphogenesis in ...

  16. Synthesis, Nicotinic Acetylcholine Receptor Binding, and Antinociceptive Properties of 3′-(Substituted Phenyl)epibatidine Analogues. Nicotinic Partial Agonists⊥

    Carroll, F. Ivy; Ma, Wei; Deng, Liu; Navarro, Hernán A.; Damaj, M. Imad; Martin, Billy R.

    2010-01-01

    In 1992, John Daly et al. reported the isolation and structure determination of epibatidine. Epibatidine’s unique structure and its potent nicotinic agonist activity have had a tremendous impact on nicotine receptor research. This research has led to a better understanding of the nicotinic acetylcholine receptor (nAChR) pharmacophore and to epibatidine analogues with potential as pharmacotherapies for treating various CNS disorders. In this study, we report the synthesis, receptor binding ([3...

  17. Immunological studies on the structure and function of the nicotinic acetylcholine receptor in mammalian muscle

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the {alpha}-bungarotoxin binding sites of the acetylcholine receptor (AChR) was examined using AChRs in the C2 mouse muscle cell line as a model. The antibodies were shown to be specific for one of the two toxin-binding sites. The effect of the antibodies in this myasthenic serum on the functional response of the receptor to cholinergic agonists was also examined using carbamylcholine-induced {sup 22}Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the {gamma}, {delta}, and {epsilon} subunit, respectively, of mammalian muscle AChRs were developed using subunit-specific synthetic peptides as antigens. Using these antibodies and monoclonal antibodies for other subunits as probes, I have identified four ({alpha}, {beta}, {gamma}, and {delta}) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the {alpha}, {beta}, and {delta} subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the {gamma}- and {epsilon}- AChRs in developing and in denervated muscles corresponds to the distribution of AChRs with slow and fast channels, respectively, and that the development changes in the channel properties of the receptor arise from a change in the subunit composition of the receptor, in which the {gamma} is replaced by {epsilon}.

  18. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications

  19. Steroids induce acetylcholine receptors on cultured human muscle: Implications for myasthenia gravis

    Kaplan, I.; Blakely, B.T.; Pavlath, G.K.; Travis, M.; Blau, H.M. (Stanford Univ. School of Medicine, CA (USA))

    1990-10-01

    Antibodies to the acetylcholine receptor (AChR), which are diagnostic of the human autoimmune disease myasthenia gravis, block AChR function and increase the rate of AChR degradation leading to impaired neuromuscular transmission. Steroids are frequently used to alleviate symptoms of muscle fatigue and weakness in patients with myasthenia gravis because of their well-documented immunosuppressive effects. The authors show here that the steroid dexamethasone significantly increases total surface AChRs on cultured human muscle exposed to myasthenia gravis sera. The results suggest that the clinical improvement observed in myasthenic patients treated with steroids is due not only to an effect on the immune system but also a direct effect on muscle. They propose that the identification and development of pharmacologic agents that augment receptors and other proteins that are reduced by human genetic or autoimmune disease will have broad therapeutic applications.

  20. Bispyridinium Compounds Inhibit Both Muscle and Neuronal Nicotinic Acetylcholine Receptors in Human Cell Lines.

    Avi Ring

    Full Text Available Standard treatment of poisoning by organophosphorus anticholinesterases uses atropine to reduce the muscarinic effects of acetylcholine accumulation and oximes to reactivate acetylcholinesterase (the effectiveness of which depends on the specific anticholinesterase, but does not directly address the nicotinic effects of poisoning. Bispyridinium molecules which act as noncompetitive antagonists at nicotinic acetylcholine receptors have been identified as promising compounds and one has been shown to improve survival following organophosphorus poisoning in guinea-pigs. Here, we have investigated the structural requirements for antagonism and compared inhibitory potency of these compounds at muscle and neuronal nicotinic receptors and acetylcholinesterase. A series of compounds was synthesised, in which the length of the polymethylene linker between the two pyridinium moieties was increased sequentially from one to ten carbon atoms. Their effects on nicotinic receptor-mediated calcium responses were tested in muscle-derived (CN21 and neuronal (SH-SY5Y cells. Their ability to inhibit acetylcholinesterase activity was tested using human erythrocyte ghosts. In both cell lines, the nicotinic response was inhibited in a dose-dependent manner and the inhibitory potency of the compounds increased with greater linker length between the two pyridinium moieties, as did their inhibitory potency for human acetylcholinesterase activity in vitro. These results demonstrate that bispyridinium compounds inhibit both neuronal and muscle nicotinic receptors and that their potency depends on the length of the hydrocarbon chain linking the two pyridinium moieties. Knowledge of structure-activity relationships will aid the optimisation of molecular structures for therapeutic use against the nicotinic effects of organophosphorus poisoning.

  1. Gamma-lactams--a novel scaffold for highly potent and selective alpha 7 nicotinic acetylcholine receptor agonists.

    Enz, Albert; Feuerbach, Dominik; Frederiksen, Mathias U; Gentsch, Conrad; Hurth, Konstanze; Müller, Werner; Nozulak, Joachim; Roy, Bernard L

    2009-03-01

    A novel class of alpha7 nicotinic acetylcholine receptor (nAChR) agonists has been discovered through high-throughput screening. The cis gamma-lactam scaffold has been optimized to reveal highly potent and selective alpha7 nAChR agonists with in vitro activity and selectivity and with good brain penetration in mice. PMID:19208472

  2. Utrophin abundance is reduced at neuromuscular junctions of patients with both inherited and acquired acetylcholine receptor deficiencies

    Slater, CR; Young, C; Wood, SJ; Bewick, GS; Anderson, LVB; Baxter, P; Fawcett, PRW; Roberts, M; Jacobson, L; Kuks, J; Vincent, A; NewsomDavis, J

    1997-01-01

    Congenital myasthenic syndromes are a heterogenous group of conditions in which muscle weakness resulting from impaired neuromuscular transmission is often present from infancy. One form of congenital myasthenic syndrome is due to a reduction of the number of acetylcholine receptors (AChRs) at the n

  3. Hippocampal α7 nicotinic acetylcholine receptor levels in patients with schizophrenia, bipolar disorder, or major depressive disorder

    Thomsen, Morten Skøtt; Weyn, Annelies; Mikkelsen, Jens D

    The α7 nicotinic acetylcholine receptor (nAChR) is involved in cognitive function and synaptic plasticity. Consequently, changes in α7 nAChR function have been implicated in a variety of mental disorders, especially schizophrenia. However, there is little knowledge regarding the levels of the α7 n...

  4. Changes in Temperature Have Opposing Effects on Current Amplitude in alpha 7 and alpha 4 beta 2 Nicotinic Acetylcholine Receptors

    Jindřichová, Marie; Lansdell, S. J.; Millar, N. S.

    2012-01-01

    Roč. 7, č. 2 (2012), e32073. E-ISSN 1932-6203 Institutional research plan: CEZ:AV0Z50110509 Keywords : effect of temperature * nicotinic acetylcholine receptor * voltage - clamp recording Subject RIV: ED - Physiology Impact factor: 3.730, year: 2012

  5. Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

    Grant, M.K.O.; Noetzel, M.J.; De Lorme, K.C.; Jakubík, Jan; Doležal, Vladimír; El-Fakahany, E. E.

    2010-01-01

    Roč. 5, č. 12 (2010), e15722-16. E-ISSN 1932-6203 R&D Projects: GA ČR GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : Xanomeline * muscarinic acetylcholine receptor Subject RIV: CE - Biochemistry Impact factor: 4.411, year: 2010

  6. Determination of anti-acetylcholine receptor antibodies in myasthenic patients by use of time-resolved fluorescence

    Říčný, Jan; Šimková, L.; Vincent, A.

    2002-01-01

    Roč. 48, č. 3 (2002), s. 549-554. ISSN 0009-9147 R&D Projects: GA MZd NF4646 Institutional research plan: CEZ:AV0Z5011922 Keywords : nicotinic acetylcholine receptor * time-resolved fluorescence method * myasthenia gravis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 4.788, year: 2002

  7. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  8. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β and δ subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the δ subunit 20-fold over basal phosphorylation and induced phosphorylation of the α subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 μM in the presence of 35 μM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of δ subunit phosphorylation was almost maximal within 5 min, whereas stimulation of α subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the δ subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the δ subunit increases the rate of AcChoR desensitization in rat myotubes

  9. A tale of two receptors: Dual roles for ionotropic acetylcholine receptors in regulating motor neuron excitation and inhibition.

    Philbrook, Alison; Barbagallo, Belinda; Francis, Michael M

    2013-07-01

    Nicotinic or ionotropic acetylcholine receptors (iAChRs) mediate excitatory signaling throughout the nervous system, and the heterogeneity of these receptors contributes to their multifaceted roles. Our recent work has characterized a single iAChR subunit, ACR-12, which contributes to two distinct iAChR subtypes within the C. elegans motor circuit. These two receptor subtypes regulate the coordinated activity of excitatory (cholinergic) and inhibitory (GABAergic) motor neurons. We have shown that the iAChR subunit ACR-12 is differentially expressed in both cholinergic and GABAergic motor neurons within the motor circuit. In cholinergic motor neurons, ACR-12 is incorporated into the previously characterized ACR-2 heteromeric receptor, which shows non-synaptic localization patterns and plays a modulatory role in controlling circuit function.(1) In contrast, a second population of ACR-12-containing receptors in GABAergic motor neurons, ACR-12GABA, shows synaptic expression and regulates inhibitory signaling.(2) Here, we discuss the two ACR-12-containing receptor subtypes, their distinct expression patterns, and functional roles in the C. elegans motor circuit. We anticipate our continuing studies of iAChRs in the C. elegans motor circuit will lead to novel insights into iAChR function in the nervous system as well as mechanisms for their regulation. PMID:24778941

  10. α7-Nicotinic acetylcholine receptor: role in early odor learning preference in mice.

    Jennifer L Hellier

    Full Text Available Recently, we have shown that mice with decreased expression of α7-nicotinic acetylcholine receptors (α7 in the olfactory bulb were associated with a deficit in odor discrimination compared to wild-type mice. However, it is unknown if mice with decreased α7-receptor expression also show a deficit in early odor learning preference (ELP, an enhanced behavioral response to odors with attractive value observed in rats. In this study, we modified ELP methods performed in rats and implemented similar conditions in mice. From post-natal days 5-18, wild-type mice were stroked simultaneously with an odor presentation (conditioned odor for 90 s daily. Control mice were only stroked, exposed to odor, or neither. On the day of testing (P21, mice that were stroked in concert with a conditioned odor significantly investigated the conditioned odor compared to a novel odor, as observed similarly in rats. However, mice with a decrease in α7-receptor expression that were stroked during a conditioned odor did not show a behavioral response to that odorant. These results suggest that decreased α7-receptor expression has a role in associative learning, olfactory preference, and/or sensory processing deficits.

  11. Brain α7 nicotinic acetylcholine receptors in MPTP-lesioned monkeys and parkinsonian patients.

    Morissette, Marc; Morin, Nicolas; Grégoire, Laurent; Rajput, Alex; Rajput, Ali H; Di Paolo, Thérèse

    2016-06-01

    L-DOPA-induced dyskinesias (LID) appear in the majority of Parkinson's disease (PD) patients. Nicotinic acetylcholine (nACh) receptor-mediated signaling has been implicated in PD and LID and modulation of brain α7 nACh receptors might be a potential therapeutic target for PD. This study used [(125)I]α-Bungarotoxin autoradiography to investigate α7 nACh receptors in LID in post-mortem brains from PD patients (n=14) and control subjects (n=11), and from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys treated with saline (n=5), L-DOPA (n=4) or L-DOPA+2-methyl-6-(phenylethynyl)pyridine (MPEP) (n=5), and control monkeys (n=4). MPEP is the prototypal metabotropic glutamate 5 (mGlu5) receptor antagonist; it reduced the development of LID in these monkeys. [(125)I]α-Bungarotoxin specific binding to striatal and pallidal α7 nACh receptors were only increased in L-DOPA-treated dyskinetic MPTP monkeys as compared to controls, saline and L-DOPA+MPEP MPTP monkeys; dyskinesia scores correlated positively with this binding. The total group of Parkinsonian patients had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus but not in the putamen. PD patients without motor complications had higher [(125)I]α-Bungarotoxin specific binding compared to controls only in the caudate nucleus. PD patients with LID only had higher [(125)I]α-Bungarotoxin specific binding compared to controls in the caudate nucleus and compared to those without motor complications and controls in the putamen. PD patients with wearing-off only, had [(125)I]α-Bungarotoxin specific binding at control values in the caudate nucleus and lower in the putamen. Reduced motor complications were associated with normal striatal α7 nACh receptors, suggesting the potential of this receptor to manage motor complications in PD. PMID:27038656

  12. Requirement of the nicotinic acetylcholine receptor β2 subunit for the anatomical and functional development of the visual system

    Rossi, Francesco Mattia; Pizzorusso, Tommaso; Porciatti, Vittorio; Marubio, Lisa M.; Maffei, Lamberto; Changeux, Jean-Pierre

    2001-01-01

    In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the β2 subunit of the neuronal nicotinic receptor, but not in mice lacking the α4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. ...

  13. Kinetics of Carbamylcholine Binding to Membrane-Bound Acetylcholine Receptor Monitored by Fluorescence Changes of a Covalently Bound Probe

    Dunn, Susan M.J.; Blanchard, Steven G.; Raftery, Michael A.

    1980-01-01

    The fluorescent probe 5-(iodoacetamido)salicylic acid has been used to alkylate acetylcholine receptor enriched membrane fragments from Torpedo californica following their reduction with low concentrations of dithiothreitol. This modification did not affect the equilibrium binding of carbamylcholine to the receptor. The fluorescence of bound 5-(iodoacetamido)salicylic acid was enhanced when the labeled membrane fragments were mixed with carbamylcholine. This increase in fluorescence was ab...

  14. Dynamic heterogeneity and non-Gaussian statistics for acetylcholine receptors on live cell membrane

    He, W.; Song, H.; Su, Y.; Geng, L.; Ackerson, B. J.; Peng, H. B.; Tong, P.

    2016-05-01

    The Brownian motion of molecules at thermal equilibrium usually has a finite correlation time and will eventually be randomized after a long delay time, so that their displacement follows the Gaussian statistics. This is true even when the molecules have experienced a complex environment with a finite correlation time. Here, we report that the lateral motion of the acetylcholine receptors on live muscle cell membranes does not follow the Gaussian statistics for normal Brownian diffusion. From a careful analysis of a large volume of the protein trajectories obtained over a wide range of sampling rates and long durations, we find that the normalized histogram of the protein displacements shows an exponential tail, which is robust and universal for cells under different conditions. The experiment indicates that the observed non-Gaussian statistics and dynamic heterogeneity are inherently linked to the slow-active remodelling of the underlying cortical actin network.

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

    Qiang LIU; Jie WU

    2006-01-01

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

  16. [3H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

    Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [3H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB2H4 (in model studies) or NaB3H4 in absolute ethanol to 2-chloro-5-pyridinylmethanol which was transformed to 2-chloro-5-chloromethylpyridine on refluxing with thionyl chloride. Coupling with 4,5-dihydro-N-nitro-1H-imidazol-2-amine then gave [2H2]imidacloprid incorporating about 95% of the deuterium or [3H2]imidacloprid (25 Ci/mmol) in 80% radiochemical yield. In studies not detailed here [3H] imidacloprid was found to undergo high affinity, specific and saturable binding to a site in insect brain. (author)

  17. Neuronal nicotinic acetylcholine receptors: Common molecular substrates of nicotine and alcohol dependence

    AndrewR.Tapper

    2013-04-01

    Full Text Available Alcohol and nicotine are often co-abused. As many as 80-95% of alcoholics are also smokers, suggesting that ethanol and nicotine, the primary addictive component of tobacco smoke, may functionally interact in the central nervous system and/or share a common mechanism of action. While nicotine initiates dependence by binding to and activating neuronal nicotinic acetylcholine receptors (nAChRs, ligand-gated cation channels normally activated by endogenous acetylcholine (ACh, ethanol is much less specific with the ability to modulate multiple gene products including those encoding voltage-gated ion channels, and excitatory/inhibitory neurotransmitter receptors. However, emerging data indicate that ethanol interacts with nAChRs, both directly and indirectly, in the mesocorticolimbic dopaminergic (DAergic reward circuitry to affect brain reward systems. Like nicotine, ethanol activates DAergic neurons of the ventral tegmental area (VTA which project to the nucleus accumbens (NAc. Blockade of VTA nAChRs reduces ethanol-mediated activation of DAergic neurons, NAc DA release, consumption, and operant responding for ethanol in rodents. Thus, ethanol may increase ACh release into the VTA driving activation of DAergic neurons through nAChRs. In addition, ethanol potentiates distinct nAChR subtype responses to ACh and nicotine in vitro and in DAergic neurons. The smoking cessation therapeutic and nAChR partial agonist, varenicline, reduces alcohol consumption in heavy drinking smokers and rodent models of alcohol consumption. Finally, single nucleotide polymorphisms in nAChR subunit genes are associated with alcohol dependence phenotypes and smoking behaviors in human populations. Together, results from preclinical, clinical, and genetic studies indicate that nAChRs may have an inherent role in the abusive properties of ethanol, as well as in nicotine and alcohol co-dependence.

  18. Design and synthesis of new agents for neuronal nicotinic acetylcholine receptor (nAChRs) imaging

    Introduction: The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Due to rapid technological progress in chemistry, bioinformatics, structural biology and computer technology, computer aided drug design (CADD) plays a more and more important role in today's drug discovery. Methods: Two novel 3-pyridyl ether nicotinic ligands-3-((pyridine-2-yl)methoxy)-5-iodopyridine, and 3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)-methyl)pyridine were designed and synthesized and radiolabeled with I-125 based on our 3D-QSAR models reported previously. Their ability to label high-affinity brain nicotinic acetylcholine receptors (nAChRs) was evaluated. Results: [125I]3-((pyridin-2-yl)methoxy)-5-iodopyridine shows rapid accumulation and elimination with peak (1.86%ID/g) at 5 min post injection, but has high blood uptake. [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine entered the brain with maximal uptake value 3.01%ID/g at 15 min after injection, and showed approximately 27% inhibition of radioactivity uptake in thalamus in mice pretreated with nicotine. Conclusions: The results of this preliminary study show that [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine shows relatively high uptake to the brain, however, since the in vivo selectivity for α4β2 nAChRs was not enough, [125I]3-(((S)-pyrrolidin-2-yl)methoxy)-5-((4-iodobenzyloxy)methyl)pyridine does not have the required properties for imaging nAChRs using SPECT. Structure optimization is needed for specific visualization of brain α4β2 nAChRs in vivo.

  19. Covalent Trapping of Methyllycaconitine at the α4-α4 Interface of the α4β2 Nicotinic Acetylcholine Receptor

    Absalom, Nathan L; Quek, Gracia; Lewis, Trevor M;

    2013-01-01

    The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their pharmacologi......The α4β2 nicotinic acetylcholine receptors (nAChRs) are widely expressed in the brain and are implicated in a variety of physiological processes. There are two stoichiometries of the α4β2 nAChR, (α4)2(β2)3 and (α4)3(β2)2, with different sensitivities to acetylcholine (ACh), but their...... competitive antagonism and an apparently insurmountable mechanism that only occurs after preincubation with MLA. We hypothesized an additional MLA binding site in the α4-α4 interface that is unique to this stoichiometry. To prove this, we covalently trapped a cysteine-reactive MLA analog at an α4β2 receptor...... containing an α4(D204C) mutation predicted by homology modeling to be within reach of the reactive probe. We demonstrate that covalent trapping results in irreversible reduction of ACh-elicited currents in the (α4)3(β2)2 stoichiometry, indicating that MLA binds to the α4-α4 interface of the (α4)3(β2)2 and...

  20. Activation of the Macrophage α7 Nicotinic Acetylcholine Receptor and Control of Inflammation.

    Báez-Pagán, Carlos A; Delgado-Vélez, Manuel; Lasalde-Dominicci, José A

    2015-09-01

    Inflammatory responses to stimuli are essential body defenses against foreign threats. However, uncontrolled inflammation may result in serious health problems, which can be life-threatening. The α7 nicotinic acetylcholine receptor, a ligand-gated ion channel expressed in the nervous and immune systems, has an essential role in the control of inflammation. Activation of the macrophage α7 receptor by acetylcholine, nicotine, or other agonists, selectively inhibits production of pro-inflammatory cytokines while leaving anti-inflammatory cytokines undisturbed. The neural control of this regulation pathway was discovered recently and it was named the cholinergic anti-inflammatory pathway (CAP). When afferent vagus nerve terminals are activated by cytokines or other pro-inflammatory stimuli, the message travels through the afferent vagus nerve, resulting in action potentials traveling down efferent vagus nerve fibers in a process that eventually leads to macrophage α7 activation by acetylcholine and inhibition of pro-inflammatory cytokines production. The mechanism by which activation of α7 in macrophages regulates pro-inflammatory responses is subject of intense research, and important insights have thus been made. The results suggest that activation of the macrophage α7 controls inflammation by inhibiting NF-κB nuclear translocation, and activating the JAK2/STAT3 pathway among other suggested pathways. While the α7 is well characterized as a ligand-gated ion channel in neurons, whole-cell patch clamp experiments suggest that α7's ion channel activity, defined as the translocation of ions across the membrane in response to ligands, is absent in leukocytes, and therefore, ion channel activity is generally assumed not to be required for the operation of the CAP. In this perspective, we briefly review macrophage α7 activation as it relates to the control of inflammation, and broaden the current view by providing single-channel currents as evidence that the α7

  1. Nicotine Ameliorates NMDA Receptor Antagonist-Induced Deficits in Contextual Fear Conditioning through High Affinity Nicotinic Acetylcholine Receptors in the Hippocampus

    André, Jessica M.; Leach, Prescott T.; Gould, Thomas J.

    2010-01-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV) induced disruption of conte...

  2. Study of acetylcholine and barium receptors in the rat duodeno-jejunum by means of labelled molecules

    The purpose of this work is the determination of the number and the localization of Acetylcholine and Barium receptors in the rat intestine. We used 'radioactive labelled' drugs to reach a high sensitiveness of detection. So we were able to point out the number of 'effective' molecules of drugs, that is to say the only ones combining with receptors. With the aid of some assumptions, we determine on the one hand the receptors localization by an assessment of the drug penetration depth before reaching their level and on the other hand the number of these receptors. (author)

  3. Stoichiometry for α-bungarotoxin block of α7 acetylcholine receptors

    Dacosta, Corrie J. B.; Free, Chris R.; Sine, Steven M.

    2015-08-01

    α-Bungarotoxin (α-Btx) binds to the five agonist binding sites on the homopentameric α7-acetylcholine receptor, yet the number of bound α-Btx molecules required to prevent agonist-induced channel opening remains unknown. To determine the stoichiometry for α-Btx blockade, we generate receptors comprised of wild-type and α-Btx-resistant subunits, tag one of the subunit types with conductance mutations to report subunit stoichiometry, and following incubation with α-Btx, monitor opening of individual receptor channels with defined subunit stoichiometry. We find that a single α-Btx-sensitive subunit confers nearly maximal suppression of channel opening, despite four binding sites remaining unoccupied by α-Btx and accessible to the agonist. Given structural evidence that α-Btx locks the agonist binding site in an inactive conformation, we conclude that the dominant mechanism of antagonism is non-competitive, originating from conformational arrest of the binding sites, and that the five α7 subunits are interdependent and maintain conformational symmetry in the open channel state.

  4. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  5. The subpopulation of microglia expressing functional muscarinic acetylcholine receptors expands in stroke and Alzheimer's disease.

    Pannell, Maria; Meier, Maria Almut; Szulzewsky, Frank; Matyash, Vitali; Endres, Matthias; Kronenberg, Golo; Prinz, Vincent; Waiczies, Sonia; Wolf, Susanne A; Kettenmann, Helmut

    2016-03-01

    Microglia undergo a process of activation in pathology which is controlled by many factors including neurotransmitters. We found that a subpopulation (11 %) of freshly isolated adult microglia respond to the muscarinic acetylcholine receptor agonist carbachol with a Ca(2+) increase and a subpopulation of similar size (16 %) was observed by FACS analysis using an antibody against the M3 receptor subtype. The carbachol-sensitive population increased in microglia/brain macrophages isolated from tissue of mouse models for stroke (60 %) and Alzheimer's disease (25 %), but not for glioma and multiple sclerosis. Microglia cultured from adult and neonatal brain contained a carbachol-sensitive subpopulation (8 and 9 %), which was increased by treatment with interferon-γ to around 60 %. This increase was sensitive to blockers of protein synthesis and correlated with an upregulation of the M3 receptor subtype and with an increased expression of MHC-I and MHC-II. Carbachol was a chemoattractant for microglia and decreased their phagocytic activity. PMID:25523105

  6. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 1010 M-1, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r2>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG

  7. Binding affinities of anti-acetylcholine receptor autoantibodies in myasthenia gravis

    Bray, J.J.; Drachman, D.B.

    1982-01-01

    Antibodies directed against acetylcholine (ACh) receptors are present in the sera of nearly 90% of patients with myasthenia gravis (MG), and are involved in the pathogenesis of this autoimmune disease. However, the antibody titers measured by the standard radioimmunoassay correspond poorly with the clinical severity of the disease. To determine whether this disparity could be accounted for by differences in the binding affinities of anti-ACh receptor antibodies in different patients, we have measured the binding affinities of these autoantibodies in 15 sera from MG patients. The affinity constants (K/sub o/), as determined by Scatchard analysis, were all in the range of 10/sup 10/ M/sup -1/, comparable to the highest values reported in immunized animals. The affinity constants were truly representative of the population of autoantibodies detected by the radioimmunoassay, as shown by the remarkable linearity of the Scatchard plots (r/sup 2/>0.90) and the close correlation between the antibody titers determined by extrapolation of the Scatchard plots and by saturation analysis (r = 0.99; p < 0.001). There was only a 6-fold variation in affinity constants measured in this series of patients despite widely differing antibody titers and severity of the disease. Factors other than the titer and affinity of anti-ACh receptor antibodies may correlate better with the clinical manifestations of MG.

  8. Muscarinic Acetylcholine Receptor Subtypes as Potential Drug Targets for the Treatment of Schizophrenia, Drug Abuse and Parkinson's Disease

    Dencker, Ditte; Thomsen, Morgane; Wörtwein, Gitta;

    2011-01-01

    's disease and drug abuse. Dopaminergic systems are regulated by cholinergic, especially muscarinic, input. Not surprisingly, increasing evidence implicates muscarinic acetylcholine receptor-mediated pathways as potential targets for the treatment of these disorders classically viewed as "dopamine based......) acetylcholine binding site. Such agents may lead to the development of novel classes of drugs useful for the treatment of psychosis, drug abuse and Parkinson's disease. The present review highlights recent studies carried out using muscarinic receptor knock-out mice and new subtype-selective allosteric ligands...... to assess the roles of M(1), M(4), and M(5) receptors in various central processes that are under strong dopaminergic control. The outcome of these studies opens new perspectives for the use of novel muscarinic drugs for several severe disorders of the CNS....

  9. Quinuclidine compounds differently act as agonists of Kenyon cell nicotinic acetylcholine receptors and induced distinct effect on insect ganglionic depolarizations.

    Mathé-Allainmat, Monique; Swale, Daniel; Leray, Xavier; Benzidane, Yassine; Lebreton, Jacques; Bloomquist, Jeffrey R; Thany, Steeve H

    2013-12-01

    We have recently demonstrated that a new quinuclidine benzamide compound named LMA10203 acted as an agonist of insect nicotinic acetylcholine receptors. Its specific pharmacological profile on cockroach dorsal unpaired median neurons (DUM) helped to identify alpha-bungarotoxin-insensitive nAChR2 receptors. In the present study, we tested its effect on cockroach Kenyon cells. We found that it induced an inward current demonstrating that it bounds to nicotinic acetylcholine receptors expressed on Kenyon cells. Interestingly, LMA10203-induced currents were completely blocked by the nicotinic antagonist α-bungarotoxin. We suggested that LMA10203 effect occurred through the activation of α-bungarotoxin-sensitive receptors and did not involve α-bungarotoxin-insensitive nAChR2, previously identified in DUM neurons. In addition, we have synthesized two new compounds, LMA10210 and LMA10211, and compared their effects on Kenyon cells. These compounds were members of the 3-quinuclidinyl benzamide or benzoate families. Interestingly, 1 mM LMA10210 was not able to induce an inward current on Kenyon cells compared to LMA10211. Similarly, we did not find any significant effect of LMA10210 on cockroach ganglionic depolarization, whereas these three compounds were able to induce an effect on the central nervous system of the third instar M. domestica larvae. Our data suggested that these three compounds could bind to distinct cockroach nicotinic acetylcholine receptors. PMID:23884575

  10. α4 nicotinic acetylcholine receptor modulated by galantamine on nigrostriatal terminals regulates dopamine receptor-mediated rotational behavior.

    Inden, Masatoshi; Takata, Kazuyuki; Yanagisawa, Daijiro; Ashihara, Eishi; Tooyama, Ikuo; Shimohama, Shun; Kitamura, Yoshihisa

    2016-03-01

    Galantamine, an acetylcholine esterase (AChE) inhibitor used to treat dementia symptoms, also acts as an allosteric potentiating ligand (APL) at nicotinic acetylcholine receptors (nAChRs). This study was designed to evaluate the allosteric effect of galantamine on nAChR regulation of nigrostrial dopaminergic neuronal function in the hemiparkinsonian rat model established by unilateral nigral 6-hydroxydopamine (6-OHDA) injection. Methamphetamine, a dopamine releaser, induced ipsilateral rotation, whereas dopamine agonists apomorphine (a non-selective dopamine receptor agonist), SKF38393 (a selective dopamine D1 receptor agonist), and quinpirole (a selective dopamine D2 receptor agonist) induced contralateral rotation. When 6-OHDA-injected rats were co-treated with nomifensine, a dopamine transporter inhibitor, a more pronounced and a remarkable effect of nicotine and galantamine was observed. Under these conditions, the combination of nomifensine with nicotine or galantamine induced the ipsilateral rotation similar to the methamphetamine-induced rotational behavior, indicating that nicotine and galantamine also induce dopamine release from striatal terminals. Both nicotine- and galantamine-induced rotations were significantly blocked by flupenthixol (an antagonist of both D1 and D2 dopamine receptors) and mecamylamine (an antagonist of nAChRs), suggesting that galantamine modulation of nAChRs on striatal dopaminergic terminals regulates dopamine receptor-mediated movement. Immunohistochemical staining showed that α4 nAChRs were highly expressed on striatal dopaminergic terminals, while no α7 nAChRs were detected. Pretreatment with the α4 nAChR antagonist dihydroxy-β-erythroidine significantly inhibited nicotine- and galantamine-induced rotational behaviors, whereas pretreatment with the α7 nAChR antagonist methyllycaconitine was ineffective. Moreover, the α4 nAChR agonist ABT-418 induced ipsilateral rotation, while the α7 nAChR agonist PNU282987 had no

  11. Acetylcholine muscarinic receptors and response to anti-cholinesterase therapy in patients with Alzheimer's disease

    An acetylcholine deficit remains the most consistent neurotransmitter abnormality found in Alzheimer's disease and various therapeutic agents have been targeted at this. In this study we investigated the action of Donepezil, a cholinesterase inhibitor that has few side-effects. In particular we set out to investigate whether muscarinic acetylcholine receptor (mAChR) availability influences the response to this therapy. We used the novel single-photon emission tomography (SPET) tracer (R,R)[123I]I-quinuclidinyl benzilate (R,R[123I]I-QNB), which has high affinity for the M1 subtype of mAChR. Regional cerebral perfusion was also assessed using technetium-99m hexamethylpropylene amine oxime. We investigated 20 patients on Donepezil treatment and ten age-matched controls. The results showed a reduction in (R,R)[123I]I-QNB binding in the caudal anterior cingulate in patients compared with controls and relatively high binding in the putamen and rostral anterior cingulate, suggesting a relative sparing of mAChR in these regions. The main finding of the study was that mAChR availability as assessed by (R,R)[123I]I-QNB binding did not distinguish responders from non-responders. Interestingly, we found that the extent of cognitive improvement showed no positive correlation with (R,R)[123I]I-QNB binding in any brain region but was inversely related to binding in the insular cortex. This suggests that, within the advised cognitive performance band for use of Donepezil, response is greater in those patients with evidence of a more marked cholinergic deficit. A larger study should investigate this. (orig.)

  12. Physiological and biochemical studies of newly synthesized muscarinic acetylcholine receptors in embryonic chicken heart

    Exposure of either chicken embryos in ovo or cultured embryonic chicken cardiac cells in vitro to the muscarinic agonist carbachol results in a 70-90% decrease in the number of muscarinic acetylcholine receptors (mAChR) expressed in cardiac cells. Block of agonist-receptor interactions in ovo with the antagonist atropine or removal of the agonist in vitro results in a gradual increase in mAChR number, reaching the control level in 14 hr. Measurements of physiological sensitivity of atria or cultured cells show that, even after the complete recovery of receptor number, the sensitivity to agonist is reduced. The sensitivity of the mAChR-mediated inhibition of adenylate cyclase is also decreased at this time. Newly synthesized mAChR which appear following affinity alkylation in cultured cells are also poorly coupled to the stimulation of 86Rb+ efflux, indicating that decreased physiological sensitivity is not due to an unknown effect of long-term agonist exposure on general cellular function, but rather reflects an intrinsic property of newly synthesized mAChR. This increase in sensitivity is also not blocked by cycloheximide. The increase in sensitivity of the mAChR-mediated responses is due neither to a lack of expression of newly synthesized mAChR on the surface nor to reduced agonist affinity of the mAChR. The diminished sensitivity and subsequent maturation observed in cells containing newly synthesized receptors is due either to a small change in mAChR, or to a change in an as-yet-undefined component of the mAChR transduction system; this alteration represents a novel locus for modulation of cholinergic signals in the heart

  13. Functional Human α7 Nicotinic Acetylcholine Receptor (nAChR) Generated from Escherichia coli.

    Tillman, Tommy S; Alvarez, Frances J D; Reinert, Nathan J; Liu, Chuang; Wang, Dawei; Xu, Yan; Xiao, Kunhong; Zhang, Peijun; Tang, Pei

    2016-08-26

    Human Cys-loop receptors are important therapeutic targets. High-resolution structures are essential for rational drug design, but only a few are available due to difficulties in obtaining sufficient quantities of protein suitable for structural studies. Although expression of proteins in E. coli offers advantages of high yield, low cost, and fast turnover, this approach has not been thoroughly explored for full-length human Cys-loop receptors because of the conventional wisdom that E. coli lacks the specific chaperones and post-translational modifications potentially required for expression of human Cys-loop receptors. Here we report the successful production of full-length wild type human α7nAChR from E. coli Chemically induced chaperones promote high expression levels of well-folded proteins. The choice of detergents, lipids, and ligands during purification determines the final protein quality. The purified α7nAChR not only forms pentamers as imaged by negative-stain electron microscopy, but also retains pharmacological characteristics of native α7nAChR, including binding to bungarotoxin and positive allosteric modulators specific to α7nAChR. Moreover, the purified α7nAChR injected into Xenopus oocytes can be activated by acetylcholine, choline, and nicotine, inhibited by the channel blockers QX-222 and phencyclidine, and potentiated by the α7nAChR specific modulators PNU-120596 and TQS. The successful generation of functional human α7nAChR from E. coli opens a new avenue for producing mammalian Cys-loop receptors to facilitate structure-based rational drug design. PMID:27385587

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

    Jian-xin SHEN; Jerrel L YAKEL

    2009-01-01

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

  15. Interaction of selective serotonin reuptake inhibitors with neuronal nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Russell, Megan; Jozwiak, Krzysztof

    2010-07-13

    We compared the interaction of fluoxetine and paroxetine, two selective serotonin reuptake inhibitors (SSRIs), with the human (h) alpha4beta2, alpha3beta4, and alpha7 nicotinic acetylcholine receptors (AChRs) in different conformational states, using Ca(2+) influx, radioligand binding, and molecular docking approaches. The results established that (1) fluoxetine was more potent than paroxetine in inhibiting agonist-activated Ca(2+) influx on halpha4beta2 and halpha7 AChRs, whereas the potency of both SSRIs was practically the same in the halpha3beta4 AChR. [corrected] (2) SSRIs bind to the [(3)H]imipramine locus with a [corrected] higher affinity when the AChRs are in the desensitized states compared to the resting states. (3) The different receptor specificity for fluoxetine determined by their inhibitory potencies or binding affinities suggests different modes of interaction when the AChR is in the closed or activated state. (4) Neutral and protonated fluoxetine interacts with a binding domain located in the middle of the AChR ion channel. In conclusion, SSRIs inhibit the most important neuronal AChRs with potencies and affinities that are clinically relevant by binding to a luminal site that is shared with tricyclic antidepressants. PMID:20527991

  16. Nicotinic acetylcholine receptor-based blockade: applications of molecular targets for cancer therapy.

    Wu, Chih-Hsiung; Lee, Chia-Hwa; Ho, Yuan-Soon

    2011-06-01

    The nicotinic acetylcholine receptor (nAChR) was first characterized in 1970 as a membrane receptor of a neurotransmitter and an ion channel. nAChRs have been shown to be involved in smoking-induced cancer formation in multiple types of human cancer cells. In vitro and in vivo animal studies have shown that homopentameric nAChR inhibitors, such as methyllycaconitine and α-Bgtx, can attenuate nicotine-induced proliferative, angiogenic, and metastatic effects in lung, colon, and bladder cancer cells. Recent publications have shown that α9-nAChR is important for breast cancer formation, and in many in vivo studies, α9-nAChR-specific antagonists (e.g., α-ImI, α-ImI, Vc1.1, RgIA, and It14a) produced an analgesic effect. Vc1.1 functions in a variety of animal pain models and currently has entered phase II clinical trials. For cancer therapy, natural compounds such as garcinol and EGCG have been found to block nicotine- and estrogen-induced breast cancer cell proliferation through inhibition of the α9-nAChR signaling pathway. A detailed investigation of the carcinogenic effects of nAChRs and their specific antagonists would enhance our understanding of their value as targets for clinical translation. PMID:21444681

  17. Thrombin action decreases acetylcholine receptor aggregate number and stability in cultured mouse myotubes.

    Davenport, R W; Lanuza, M; Kim, S; Jia, M; Snyder, E; Nelson, P G

    2000-08-30

    Neurons develop and make very stable, long-term synaptic connections with other nerve cells and with muscle. Synaptic stability at the neuromuscular junction changes over development in that a proliferation of synaptic input are made to individual myotubes and synapses from all but one neuron are lost during development. In an established co-culture paradigm in which spinal motoneurons synaptically contact myotubes, thrombin and associated protease inhibitors have been shown to affect the loss of functional synaptic contacts [6]. Evidence has not been provided which clearly demonstrate whether protease/protease inhibitors affect either the pre- or postsynaptic terminal, or both. In an effort to determine whether these reagents directly affect postsynaptic receptors on myotubes, myotubes were cultured in the absence of neurons and the spontaneous presence and stability of aggregates of acetylcholine receptors (AChR) in control and thrombin-containing media were evaluated. In dishes fixed after treatment and in dishes in which individual aggregates were observed live, thrombin action appeared to increase loss of AChR aggregates over time. Hirudin, a specific inhibitor of the thrombin protease, diminished this loss. Neither reagent affected the overall incorporation or degradation of AChR; therefore, it appears these protease/protease inhibitors affect the state of AChR aggregation. PMID:10960680

  18. Synthesis of carbon-11 labeled dexetimide and levetimide for studying muscarinic acetylcholine receptors

    The localization and quantitation of the muscarinic acetylcholine receptor (m-AChR) in the living human brain using a non-invasive method, such as positron emission tomography (PET), may provide valuable information about receptor changes which have been observed post mortem in patients with Huntington's chorea and Alzheimer's dementia, as well as normal brain mechanisms mediated by the m-AChR. Although quinuclidinyl benzilate has been radioiodinated and radiomethylatd, the former is not useful with PET and the latter does not penetrate the blood-brain barrier; therefore, the authors chose to radiolabel dexetimide, a ligand which labels m-AChR in vitro and in vivo, and levetimide, its inactive enantiomer. Carbon-11 labeled carbon dioxide is bubbled through a tetrahydrofuran (THF) solution of phenylmagnesium chloride (1 M, l ml) after which 2 mg of lithium aluminium hydride is added in THF (500 μl). After evaporation of the solvent, 48% hydriodic acid (l ml) is added and the solution is heated for 1 minute. Carbon-11 labeled benzyl iodide is extracted into methylene chloride, added to a solution of nor-benzyl dexetimide or levetimide, and heated for several minutes. Purification is accomplished using semi-preparative reverse phase high performance liquid chromatography (HPLC). Analytical HPLC is used to determine the radiochemical purity and specific activity

  19. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    Supavilai, P.; Karobath, M.

    1985-02-04

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with /sup 3/H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 ..mu..M. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table.

  20. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex

    GABA, THIP and muscimol enhance spontaneous and inhibit electrically induced release of tritium labelled compounds from rat striatal slices which have been pre-labelled with 3H-choline. Baclofen is inactive in this model. Muscimol can inhibit electrically induced release of tritiated material by approximately 75% with half maximal effects at 2 μM. The response to muscimol can be blocked by the GABA antagonists bicuculline methobromide, picrotoxin, anisatin, R 5135 and CPTBO (cyclopentylbicyclophosphate). Drugs which act on the benzodiazepine receptor (BR) require the presence of muscimol to be effective and they modulate the effects of muscimol in a bidirectional manner. Thus BR agonists enhance and inverse BR agonists attenuate the inhibitory effects of muscimol on electrically induced release. Ro15-1788, a BR antagonist, does not modulate the inhibitory effects of muscimol but antagonizes the actions of clonazepam, a BR agonist, and of DMCM, an inverse BR agonist. These results demonstrate that a GABA/benzodiazepine receptor complex can modulate acetylcholine release from rat striatal slices in vitro. 24 references, 3 figures, 5 table

  1. Gating of long-term potentiation by nicotinic acetylcholine receptors at the cerebellum input stage.

    Francesca Prestori

    Full Text Available The brain needs mechanisms able to correlate plastic changes with local circuit activity and internal functional states. At the cerebellum input stage, uncontrolled induction of long-term potentiation or depression (LTP or LTD between mossy fibres and granule cells can saturate synaptic capacity and impair cerebellar functioning, which suggests that neuromodulators are required to gate plasticity processes. Cholinergic systems innervating the cerebellum are thought to enhance procedural learning and memory. Here we show that a specific subtype of acetylcholine receptors, the α7-nAChRs, are distributed both in cerebellar mossy fibre terminals and granule cell dendrites and contribute substantially to synaptic regulation. Selective α7-nAChR activation enhances the postsynaptic calcium increase, allowing weak mossy fibre bursts, which would otherwise cause LTD, to generate robust LTP. The local microperfusion of α7-nAChR agonists could also lead to in vivo switching of LTD to LTP following sensory stimulation of the whisker pad. In the cerebellar flocculus, α7-nAChR pharmacological activation impaired vestibulo-ocular-reflex adaptation, probably because LTP was saturated, preventing the fine adjustment of synaptic weights. These results show that gating mechanisms mediated by specific subtypes of nicotinic receptors are required to control the LTD/LTP balance at the mossy fibre-granule cell relay in order to regulate cerebellar plasticity and behavioural adaptation.

  2. A Subpopulation of Neuronal M4 Muscarinic Acetylcholine Receptors Plays a Critical Role in Modulating Dopamine-Dependent Behaviors

    Jeon, Jongrye; Dencker, Ditte; Wortwein, Gitta; Woldbye, David P.D.; Cui, Yinghong; Davis, Albert A.; Levey, Allan I.; Schütz, Günther; Sager, Thomas; Mørk, Arne; Li, Cuiling; Deng, Chu-Xia; Fink-Jensen, Anders; Wess, Jürgen

    2010-01-01

    Acetylcholine (ACh) regulates many key functions of the CNS by activating cell surface receptors referred to as muscarinic ACh receptors (M1–M5 mAChRs). Like other mAChR subtypes, the M4 mAChR is widely expressed in different regions of the forebrain. Interestingly, M4 mAChRs are coexpressed with D1 dopamine receptors in a specific subset of striatal projection neurons. To investigate the physiological relevance of this M4 mAChR subpopulation in modulating dopamine-dependent behaviors, we use...

  3. Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments

    Asmar-Rovira, Guillermo A.; Asseo-García, Aloysha M.; Quesada, Orestes; Hanson, Michael A.; Nogueras, Carlos; Lasalde-Dominicci, José A.; Stevens, Raymond C.

    2008-01-01

    The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, the molecular mechanisms by which detergents influence membrane protein stability and function remain poorly understood, and elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examined nine detergents upon nAChR solubilization and purification, to assess re...

  4. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    Kasheverov, Igor E.; Irina V. Shelukhina; Kudryavtsev, Denis S.; Tatyana N. Makarieva; Spirova, Ekaterina N.; Alla G. Guzii; Stonik, Valentin A.; Tsetlin, Victor I.

    2015-01-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of ch...

  5. Effects of extracts from Cordyceps sinensis on M1 muscarinic acetylcholine receptor in vitro and in vivo

    Chiba, Tomohiro

    2010-01-01

    Tomohiro Chiba1, Marina Yamada1, Kosuke Torii2, Masataka Suzuki1, Jumpei Sasabe1, Minoru Ito2, Kenzo Terashita1, Sadakazu Aiso11Department of Anatomy, Keio University, School of Medicine, Tokyo, Japan; 2Department of Research and Development, Noevir Co. Ltd., Tokyo, JapanAbstract: Cholinergic dysfunction is implicated in the pathogenesis of memory impairment related to Alzheimer’s disease (AD). Accordingly, regulation of M1 muscarinic acetylcholine receptor (M1 mAChR) has been one o...

  6. Nicotinic Acetylcholine Receptor Agonists Attenuate Septic Acute Kidney Injury in Mice by Suppressing Inflammation and Proteasome Activity

    Chatterjee, Prodyot K.; Yeboah, Michael M.; Oonagh Dowling; Xiangying Xue; Powell, Saul R.; Yousef Al-Abed; Metz, Christine N

    2012-01-01

    Sepsis is one of the leading causes of acute kidney injury (AKI). Septic patients who develop acute kidney injury (AKI) are at increased risk of death. To date there is no effective treatment for AKI or septic AKI. Based on their anti-inflammatory properties, we examined the effects of nicotinic acetylcholine receptor agonists on renal damage using a mouse model of lipopolysaccharide (LPS)-induced AKI where localized LPS promotes inflammation-mediated kidney damage. Administration of nicotine...

  7. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases

  8. Thymus cells in myasthenia gravis selectively enhance production of anti-acetylcholine-receptor antibody by autologous blood lymphocytes

    Newsom-Davis, J.; Willcox, N.; Calder, L.

    1981-11-26

    We investigated the role of the thymus in 16 patients with myasthenia gravis without thymoma by studying the production of anti-acetylcholine-receptor antibody by thymic and blood lymphocytes cultured alone or together. In 10 responders (with the highest receptor-antibody titers in their plasma), cultured thymic cells spontaneously produced measurable receptor antibody. Receptor-antibody production by autologous blood lymphocytes was enhanced by the addition of responder's thymic cells, irradiated to abrogate antibody production and suppression (P<0.01). This enhancement was greater and more consistent than that by pokeweed mitogen; it depended on viable thymic cells, appeared to be selective for receptor antibody, and correlated with the ratio of thymic helper (OKT4-positive or OKT4+) to suppressor (OKT8+) T cells (P<0.01). These results suggest that myasthenic thymus contains cell-bound acetylcholine-receptor-like material or specific T cells (or both) that can aid receptor-antibody production. This may be relevant to the benefits of thymectomy in myasthenia and to the breakdown in self-tolerance in this and other autoimmune diseases.

  9. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    Highlights: ► Cigarette smoke may induce liver fibrosis via nicotine receptors. ► Nicotine induces proliferation of hepatic stellate cells (HSCs). ► Nicotine activates hepatic fibrogenic pathways. ► Nicotine receptor antagonists attenuate HSC proliferation. ► Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine – which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed – RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-α2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type (α1, β1, delta and epsilon) and neuronal type (α3, α6, α7, β2 and β4) nAChR subunits at the mRNA level. Among these subunits, α3, α7, β1 and ε were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-α2 and TGF-β1 mRNA expression were significantly upregulated by nicotine and inhibited by mecamylamine. α1 and α3-nAChR mRNA expression was significantly upregulated in NASH fibrosis compared to normal livers. Conclusion: Nicotine at levels in smokers’ blood is pro-fibrogenic, through

  10. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  11. Interaction of bupropion with muscle-type nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Gumilar, Fernanda; Rosenberg, Avraham; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W; Bouzat, Cecilia

    2009-06-01

    To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion (a) inhibits epibatidine-induced Ca(2+) influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with approximately 100-fold higher potency compared to that in the open state, (c) increases the desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits binding of [(3)H]TCP and [(3)H]imipramine to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/alpha-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy-driven process, and (f) interacts with a binding domain located between the serine (position 6') and valine (position 13') rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively, our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process. PMID:19334677

  12. Interaction of Bupropion with Muscle-Type Nicotinic Acetylcholine Receptors in Different Conformational States†

    Arias, Hugo R.; Gumilar, Fernanda; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.; Bouzat, Cecilia

    2009-01-01

    To characterize the binding sites and the mechanisms of inhibition of bupropion on muscle-type nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The results established that bupropion: (a) inhibits epibatidine-induced Ca2+ influx in embryonic muscle AChRs, (b) inhibits adult muscle AChR macroscopic currents in the resting/activatable state with ~100-fold higher potency compared to that in the open state, (c) increases desensitization rate of adult muscle AChRs from the open state and impairs channel opening from the resting state, (d) inhibits [3H]TCP and [3H]imipramine binding to the desensitized/carbamylcholine-bound Torpedo AChR with higher affinity compared to the resting/α-bungarotoxin-bound AChR, (e) binds to the Torpedo AChR in either state mainly by an entropy–driven process, and (f) interacts with a binding domain located between the serine (position 6’) and valine (position 13’) rings, by a network of van der Waals, hydrogen bond, and polar interactions. Collectively our data indicate that bupropion first binds to the resting AChR, decreasing the probability of ion channel opening. The remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process. Bupropion interacts with a luminal binding domain shared with PCP that is located between the serine and valine rings, and this interaction is mediated mainly by an entropy-driven process. PMID:19334677

  13. Alpha9 alpha10 nicotinic acetylcholine receptors as target for the treatment of chronic pain.

    Del Bufalo, Alessandra; Cesario, Alfredo; Salinaro, Gianluca; Fini, Massimo; Russo, Patrizia

    2014-01-01

    Chronic pain is a widespread healthcare problem affecting not only the patient but in many ways all the society. Chronic pain is a disease itself that endures for a long period of time and it is resistant to the majority of medical treatments that provide modest improvements in pain and minimum improvements in physical and emotional functioning. More co-existing chronic pain conditions may be present in the same individual (patient). The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in the pathophysiology of chronic pain, as well in the development of breast and lung cancers. α-conotoxins (α-CNT) are small peptides used offensively by carnivorous marine snails known as Conus that target nAChR. Among α-CNT there are potent and selective antagonists of α9α10 nAChR such as RgIA and Vc1.1 that produces both acute and long lasting analgesia. Moreover, these peptides accelerate the recovery of nerve function after injury, likely through immune/inflammatory-mediated mechanisms. We review the background, findings, implications and problems in using compounds that act on α9α10 nAChR. PMID:24641230

  14. Neonicotinoids target distinct nicotinic acetylcholine receptors and neurons, leading to differential risks to bumblebees

    Moffat, Christopher; Buckland, Stephen T.; Samson, Andrew J.; McArthur, Robin; Chamosa Pino, Victor; Bollan, Karen A.; Huang, Jeffrey T.-J.; Connolly, Christopher N.

    2016-04-01

    There is growing concern over the risk to bee populations from neonicotinoid insecticides and the long-term consequences of reduced numbers of insect pollinators to essential ecosystem services and food security. Our knowledge of the risk of neonicotinoids to bees is based on studies of imidacloprid and thiamethoxam and these findings are extrapolated to clothianidin based on its higher potency at nicotinic acetylcholine receptors. This study addresses the specificity and consequences of all three neonicotinoids to determine their relative risk to bumblebees at field-relevant levels (2.5 ppb). We find compound-specific effects at all levels (individual cells, bees and whole colonies in semi-field conditions). Imidacloprid and clothianidin display distinct, overlapping, abilities to stimulate Kenyon cells, indicating the potential to differentially influence bumblebee behavior. Bee immobility was induced only by imidacloprid, and an increased vulnerability to clothianidin toxicity only occurred following chronic exposure to clothianidin or thiamethoxam. At the whole colony level, only thiamethoxam altered the sex ratio (more males present) and only clothianidin increased queen production. Finally, both imidacloprid and thiamethoxam caused deficits in colony strength, while no detrimental effects of clothianidin were observed. Given these findings, neonicotinoid risk needs to be considered independently for each compound and target species.

  15. Neuronal Acetylcholine Nicotinic Receptors as New Targets for Lung Cancer Treatment.

    Mucchietto, Vanessa; Crespi, Arianna; Fasoli, Francesca; Clementi, Francesco; Gotti, Cecilia

    2016-01-01

    Lung cancer is the leading cause of cancer-related deaths worldwide. Smoking accounts for approximately 70% of the cases of non- small cell lung cancer (NSCLC) and 90% of the cases of small-cell lung cancer (SCLC), although some patients develop lung cancer without a history of smoking. Nicotine is the most active addictive component of tobacco smoke. It does not initiate tumorigenesis in humans and rodents, but it alters the pathophysiology of lung cells by inducing the secretion of growth factors, neurotransmitters and cytokines, and promotes tumour growth and metastases by inducing cell cycle progression, migration, invasion, angiogenesis and the evasion of apoptosis. Most of these effects are a result of nicotine binding and activation of cell-surface neuronal nicotinic acetylcholine receptors (nAChRs) and downstream intracellular signalling cascades, and many are blocked by nAChR subtype-selective antagonists. Recent genome-wide association studies have revealed single nucleotide polymorphisms of nAChR subunits that influence nicotine dependence and lung cancer. This review describes the molecular basis of nAChR structural and functional diversity in normal and cancer lung cells, and the genetic alterations facilitating smoking-induced lung cancers. It also summarises current knowledge concerning the intracellular pathways activated by nicotine and other compounds present in tobacco smoke. PMID:26845123

  16. Different patterns of nicotinic acetylcholine receptor subunit transcription in human thymus.

    Bruno, Roxana; Sabater, Lidia; Tolosa, Eva; Sospedra, Mireia; Ferrer-Francesch, Xavier; Coll, Jaume; Foz, Marius; Melms, Arthur; Pujol-Borrell, Ricardo

    2004-04-01

    Clinical observations suggest that the thymus is strongly implicated in the pathogenesis of myasthenia gravis (MG), but questions such as the level and location of nicotinic acetylcholine receptor (AChR) subunit expression that are fundamental to postulate any pathogenic mechanism, remain controversial. We have re-examined this question by combining calibrated RT-PCR and real-time PCR to study nicotinic AChR subunit mRNA expression in a panel of normal and myasthenic thymi. The results suggest that the expression of the different AChR subunits follows three distinct patterns: constitutive for, neonatal for gamma and individually variable for alpha1, beta1 and delta. Experiments using confocal laser microdissection suggest that AChR is mainly expressed in the medullary compartment of the thymus but there is not a clear compartmentalization of subunit expression. The different patterns of subunit expression may influence decisively the level of central tolerance to the subunits and explain the focusing of the T cell response to the alpha and gamma subunits. PMID:15020075

  17. Catharanthine alkaloids are noncompetitive antagonists of muscle-type nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2010-09-01

    We compared the interaction of several catharanthine alkaloids including, ibogaine, vincristine, and vinblastine, with that for the noncompetitive antagonist phencyclidine (PCP) at muscle nicotinic acetylcholine receptors (AChRs) in different conformational states. The results established that catharanthine alkaloids: (a) inhibit, in a noncompetitive manner, (+/-)-epibatidine-induced Ca(2+) influx in TE671-halpha1beta1gammadelta cells with similar potencies (IC(50)=17-25microM), (b) inhibit [(3)H]TCP binding to the desensitized Torpedo AChR with higher affinity compared to the resting AChR, and (c) enhance [(3)H]cytisine binding to resting but activatable Torpedo AChRs, suggesting desensitizing properties. Interestingly, PCP inhibits [(3)H]ibogaine binding to the AChR in a steric fashion. This is corroborated by additional docking experiments indicating that the amino groups of neutral ibogaine form hydrogen bonds with the serine ring (position 6'), a location shared with PCP. Since protonated ibogaine forms a salt bridge with one of the acidic residues at the outer ring (position 20'), this ligand could be first attracted to the entrance of the channel by electrostatic interactions. Our data indicate that the catharanthine moiety is a minimum structural requirement for AChR inhibition including, ion channel blocking and desensitization, and that ibogaine and PCP bind to overlapping sites in the desensitized AChR ion channel. PMID:20493225

  18. Interaction of 18-methoxycoronaridine with nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Rosenberg, Avraham; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Maciejewski, Ryszard; Jozwiak, Krzysztof; Moaddel, Ruin; Glick, Stanley D; Wainer, Irving W

    2010-06-01

    The interaction of 18-methoxycoronaridine (18-MC) with nicotinic acetylcholine receptors (AChRs) was compared with that for ibogaine and phencyclidine (PCP). The results established that 18-MC: (a) is more potent than ibogaine and PCP inhibiting (+/-)-epibatidine-induced AChR Ca(2+) influx. The potency of 18-MC is increased after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs, (b) binds to a single site in the Torpedo AChR with high affinity and inhibits [(3)H]TCP binding to desensitized AChRs in a steric fashion, suggesting the existence of overlapping sites. This is supported by our docking results indicating that 18-MC interacts with a domain located between the serine (position 6') and valine (position 13') rings, and (c) inhibits [(3)H]TCP, [(3)H]ibogaine, and [(3)H]18-MC binding to desensitized AChRs with higher affinity compared to resting AChRs. This can be partially attributed to a slower dissociation rate from the desensitized AChR compared to that from the resting AChR. The enthalpic contribution is more important than the entropic contribution when 18-MC binds to the desensitized AChR compared to that for the resting AChR, and vice versa. Ibogaine analogs inhibit the AChR by interacting with a luminal domain that is shared with PCP, and by inducing desensitization. PMID:20303928

  19. Neuronal Nicotinic Acetylcholine Receptors: Neuroplastic Changes underlying Alcohol and Nicotine Addictions

    Allison Anne Feduccia

    2012-08-01

    Full Text Available Addictive drugs can activate systems involved in normal reward-related learning, creating long-lasting memories of the drug’s reinforcing effects and the environmental cues surrounding the experience. These memories significantly contribute to the maintenance of compulsive drug use as well as cue-induced relapse which can occur even after long periods of abstinence. Synaptic plasticity is thought to be a prominent molecular mechanism underlying drug-induced learning and memories. Ethanol and nicotine are both widely abused drugs that share a common molecular target in the brain, the neuronal nicotinic acetylcholine receptors (nAChRs. The nAChRs are ligand-gated ion channels that are vastly distributed throughout the brain and play a key role in synaptic neurotransmission. In this review, we will delineate the role of nAChRs in the development of ethanol and nicotine addiction. We will characterize both ethanol and nicotine’s effects on nAChR-mediated synaptic transmission and plasticity in several key brain areas that are important for addiction. Finally, we will discuss some of the behavioral outcomes of drug-induced synaptic plasticity in animal models. An understanding of the molecular and cellular changes that occur following administration of ethanol and nicotine will lead to better therapeutic strategies.

  20. Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular junction by muscle activity

    The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 125I-alpha-bungarotoxin (alpha-butx). The results show that the metabolic stability of the AChRs in ectopic clusters is reversibly increased by muscle activity even when innervation is eliminated very early in development. 1 d of stimulation is sufficient to stabilize the AChRs in ectopic AChR clusters. Muscle stimulation also produced an increase in the number of AChRs at early denervated end plates. Activity-induced cluster growth occurs mainly by an increase in area rather than in AChR density, and for at least 10 d after denervation is comparable to that in normally developing ectopic end plates. The possible involvement of AChR stabilization in end plate growth is discussed

  1. Synthesis and 125I labelling of a precursor for imaging nicotinic acetylcholine receptors

    Nicotinic Acetylcholine Receptors (nAChRs) are involved in various pharmacological effects or diseases, such as Alzheimer's Disease, Parkinson's Disease and tobacco addiction. It will be very appealing to image nAChRs in vivo, diagnose and treat the above diseases, and probe the mechanism of nAChRs in tobacco addiction if the suitable radioactive labeled compound can be synthesized. In this study, (s)-5-(tri-butylstannyl)-3{[1-(tert-butoxycarbonyl)-2-azetidinyl]methoxy} pyridine, a precursor for imaging nAChRs, was synthesized with commercial 2-furfurylamine and (s)-2-azetidinecarboxylic acid as starting materials, and was further labeled with 125/123I. The whole procedure for radiosynthesis needs 50-55 min and more than 30% of the 125I are found in the purified 5-[125I]-A-85380. Even staying for 3 days at room temperature in vitro, the purified 5-[125I]-I-85380 can maintain its stability, with a radiochemical purity of more than 95%. (authors)

  2. A radioimmunoassay for the quantitative evaluation of anti-human acetylcholine receptor antibodies in myasthenia gravis

    A radioimmunoassay was developed for the quantitative evaluation of antibodies to the acetylcholine receptor in the serum of myasthenic patients. AcChR was extracted from human muscle. A detailed preparation of the 125I-labelled α-Bgt-AcChR complex used as antigen is reported. Usually, an average of 20 pmol were obtained from 100 g muscle. This preparation is stable for 1 month in presence of an inhibitor of proteolysis and sufficient for performing about fifteen assays. The labelled complex was incubated with increasing amounts of sera and precipitated with anti-human IgG serum. Titres were expressed in pmol 125I-labelled α-Bgt-AcChR complex precipitated per ml serum. Out of thirty-nine sera tested thirty-six had positive titres ranging from 0.1 to 46 pmol/ml. No anti-AcChR were detected in the sera from twenty-seven patients used as controls. (author)

  3. Nicotinic Acetylcholine Receptor α4 Subunit Gene Variation Associated with Attention Deficit Hyperactivity Disorder

    HUANG Xuezhu; XU Yong; LI Qianqian; LIU Pozi; YANG Yuan; ZHANG Fuquan; GUO Tianyou; YANG Chuang; GUO Lanting

    2009-01-01

    Previous pharmacological, human genetics, and animal models have implicated the nicotinic ace-tylcholine receptor a4 subunit (CHRNA4) gene in the pathogenesis of attention deficit/hyperactivity disorder (ADHD). The objective of this study is to examine the genetic association between single nucleotide poly-morphisms in the CHRNA4 gene (rs2273502, rs1044396, rs1044397, and rs3827020 loci) and ADHD. Both case-control and family-based designs are used. Children aged 6 to 16 years were interviewed and as-sessed with the children behavior checklist and the revised conner' parent rating scale to identify probands. No significant differences in the frequency distribution of genotypes or alleles were found between the case and control groups. However, further haplotype analyses showed the CCGG haplotype on dsk for ADHD in 164 case-control samples and the standard transmission disequilibrium test analyses suggest that the allele C of rs2273502 was over-transferred in 98 ADHD parent-offspring tdos. These findings suggest that the CHRNA4 gene may play a role in the pathogenesis of ADHD.

  4. Functional nicotinic acetylcholine receptor reconstitution in Au(111)-supported thiolipid monolayers

    Pissinis, Diego E.; Diaz, Carolina; Maza, Eliana; Bonini, Ida C.; Barrantes, Francisco J.; Salvarezza, Roberto C.; Schilardi, Patricia L.

    2015-09-01

    The insertion and function of the muscle-type nicotinic acetylcholine receptor (nAChR) in Au(111)-supported thiolipid self-assembled monolayers have been studied by atomic force microscopy (AFM), surface plasmon resonance (SPR), and electrochemical techniques. It was possible for the first time to resolve the supramolecular arrangement of the protein spontaneously inserted in a thiolipid monolayer in an aqueous solution. Geometric supramolecular arrays of nAChRs were observed, most commonly in a triangular form compatible with three nAChR dimers of ~20 nm each. Addition of the full agonist carbamoylcholine activated and opened the nAChR ion channel, as revealed by the increase in capacitance relative to that of the nAChR-thiolipid system under basal conditions. Thus, the self-assembled system appears to be a viable biomimetic model to measure ionic conductance mediated by ion-gated ion channels under different experimental conditions, with potential applications in biotechnology and pharmacology.

  5. Multiple Transmembrane Binding Sites for p-Trifluoromethyldiazirinyl-etomidate, a Photoreactive Torpedo Nicotinic Acetylcholine Receptor Allosteric Inhibitor*

    Hamouda, Ayman K.; Stewart, Deirdre S.; Husain, S. Shaukat; Cohen, Jonathan B.

    2011-01-01

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [3H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[3H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our unders...

  6. (-)-Reboxetine inhibits muscle nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

    Arias, Hugo R; Ortells, Marcelo O; Feuerbach, Dominik

    2013-11-01

    The interaction of (-)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (-)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca(2+) influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50=3.86±0.49 and 1.92±0.48 μM, respectively, (b) binds to the [(3)H]TCP site with ~13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [(3)H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6' and 13' in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9' is the minimum structural component for (-)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (-)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. PMID:23917086

  7. Propofol and AZD3043 Inhibit Adult Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

    Malin Jonsson Fagerlund

    2016-02-01

    Full Text Available Propofol is a widely used general anaesthetic with muscle relaxant properties. Similarly as propofol, the new general anaesthetic AZD3043 targets the GABAA receptor for its anaesthetic effects, but the interaction with nicotinic acetylcholine receptors (nAChRs has not been investigated. Notably, there is a gap of knowledge about the interaction between propofol and the nAChRs found in the adult neuromuscular junction. The objective was to evaluate whether propofol or AZD3043 interact with the α1β1δε, α3β2, or α7 nAChR subtypes that can be found in the neuromuscular junction and if there are any differences in affinity for those subtypes between propofol and AZD3043. Human nAChR subtypes α1β1δε, α3β2, and α7 were expressed into Xenopus oocytes and studied with an automated voltage-clamp. Propofol and AZD3043 inhibited ACh-induced currents in all of the nAChRs studied with inhibitory concentrations higher than those needed for general anaesthesia. AZD3043 was a more potent inhibitor at the adult muscle nAChR subtype compared to propofol. Propofol and AZD3043 inhibit nAChR subtypes that can be found in the adult NMJ in concentrations higher than needed for general anaesthesia. This finding needs to be evaluated in an in vitro nerve-muscle preparation and suggests one possible explanation for the muscle relaxant effect of propofol seen during higher doses.

  8. Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

    Yi Miao

    2013-02-01

    Full Text Available Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS by measuring mitochondrial ROS (mtROS, mitochondrial DNA (mtDNA copy number, xanthine oxidase (XO and NADPH oxidase (NOX activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

  9. Influence of Y151 F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

    Feng Song; Yi-Xi Zhang; Xiang-Mei Yao; Ze-Wen Liu

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels,which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems.The nAChR agonist-binding site is present at the interface of adjacent subunits and is formed by loops A-C present in α subunits together with loops D-F present in either non-α subunits or homomer-forrning α subunits.Although Y151 in loop B has been identified as important in agonist binding,various residues at the 151-site are found among vertebrate and invertebrate nAChR ot subunits,such as F151.In Xenopus oocytes expressing N1α1 or N1α1~(Y151F) plus rat β2,Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine,but not imidacloprid,including the decrease of I_(max),the increase of EC_(50)(the concentration causing 50% of the maximum response) and the fast time-constant of decay (τ_f).By comparisons of residue structure,the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between N1α1/β2 nAChRs and acetylcholine,and the phenyl group to be important between N1α1/β2 nAChRs and imidacloprid.

  10. Synergistic effect between 5-HT4 receptor agonist and phosphodiesterase 4-inhibitor in releasing acetylcholine in pig gastric circular muscle in vitro.

    Lefebvre, Romain A; Van Colen, Inge; Pauwelyn, Vicky; De Maeyer, Joris H

    2016-06-15

    5-HT4 receptor agonists have a gastroprokinetic effect by facilitating acetylcholine release from cholinergic nerves innervating gastrointestinal smooth muscle. The role of phosphodiesterase (PDE) 4 in the signal transduction pathway of the 5-HT4 receptors located on the cholinergic neurons towards the circular muscle layer in pig stomach was investigated by analysis of acetylcholine release. Circular muscle strips were prepared from pig proximal stomach and tritium outflow, induced by electrical field stimulation, was studied as a marker for acetylcholine release after incubation with [(3)H]-choline. The PDE4-inhibitor roflumilast concentration-dependently (0.1-1µM) enhanced the facilitating effect of a submaximally effective concentration of the 5-HT4 receptor agonist prucalopride (0.01µM) on electrically induced acetylcholine release. Roflumilast (0.3µM) enhanced acetylcholine release per se but in the combined presence of roflumilast and prucalopride, acetylcholine release was enhanced more than the sum of the effect of the 2 compounds alone. The 5-HT4 receptor agonist velusetrag concentration-dependently (0.01-0.1µM) enhanced acetylcholine release; the effect of the minimally effective concentration (0.01µM) was significantly enhanced by 1µM of the PDE4-inhibitor rolipram, again to a level higher than the sum of the effect of the 2 compounds alone. The synergistic effect between 5-HT4 receptor agonists and PDE4-inhibitors demonstrates that the intracellular pathway of the 5-HT4 receptors located on cholinergic neurons towards pig gastric circular muscle is controlled by PDE4. Combining a 5-HT4 receptor agonist with a PDE4-inhibitor might thus enhance its gastroprokinetic effect. PMID:27060014

  11. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-08-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs.

  12. Secreted Isoform of Human Lynx1 (SLURP-2): Spatial Structure and Pharmacology of Interactions with Different Types of Acetylcholine Receptors

    Lyukmanova, E. N.; Shulepko, M. A.; Shenkarev, Z. O.; Bychkov, M. L.; Paramonov, A. S.; Chugunov, A. O.; Kulbatskii, D. S.; Arvaniti, M.; Dolejsi, Eva; Schaer, T.; Arseniev, A. S.; Efremov, R. G.; Thomsen, M. S.; Dolezal, V.; Bertrand, D.; Dolgikh, D. A.; Kirpichnikov, M. P.

    2016-01-01

    Human-secreted Ly-6/uPAR-related protein-2 (SLURP-2) regulates the growth and differentiation of epithelial cells. Previously, the auto/paracrine activity of SLURP-2 was considered to be mediated via its interaction with the α3β2 subtype of the nicotinic acetylcholine receptors (nAChRs). Here, we describe the structure and pharmacology of a recombinant analogue of SLURP-2. Nuclear magnetic resonance spectroscopy revealed a ‘three-finger’ fold of SLURP-2 with a conserved β-structural core and three protruding loops. Affinity purification using cortical extracts revealed that SLURP-2 could interact with the α3, α4, α5, α7, β2, and β4 nAChR subunits, revealing its broader pharmacological profile. SLURP-2 inhibits acetylcholine-evoked currents at α4β2 and α3β2-nAChRs (IC50 ~0.17 and >3 μM, respectively) expressed in Xenopus oocytes. In contrast, at α7-nAChRs, SLURP-2 significantly enhances acetylcholine-evoked currents at concentrations <1 μM but induces inhibition at higher concentrations. SLURP-2 allosterically interacts with human M1 and M3 muscarinic acetylcholine receptors (mAChRs) that are overexpressed in CHO cells. SLURP-2 was found to promote the proliferation of human oral keratinocytes via interactions with α3β2-nAChRs, while it inhibited cell growth via α7-nAChRs. SLURP-2/mAChRs interactions are also probably involved in the control of keratinocyte growth. Computer modeling revealed possible SLURP-2 binding to the ‘classical’ orthosteric agonist/antagonist binding sites at α7 and α3β2-nAChRs. PMID:27485575

  13. Analogues of neuroactive polyamine wasp toxins that lack inner basic sites exhibit enhanced antagonism toward a muscle-type mammalian nicotinic acetylcholine receptor

    Stromgaard, K; Brierley, M J; Andersen, K; Sløk, F A; Mellor, I R; Usherwood, P N; Krogsgaard-Larsen, P; Jaroszewski, J W

    1999-01-01

    noncompetitively antagonized the nicotinic acetylcholine receptor (nAChR) in a concentration-, time-, and voltage-dependent manner. The amplitudes of acetylcholine-induced currents were compared at their peaks and at the end of a 1 s application in the presence or absence of the analogues. Most of the analogues...... properties (stepwise macroscopic pK(a) values) were determined by (13)C NMR titrations. All analogues are fully protonated at physiological pH. The effects of these compounds on acetylcholine-induced currents in TE671 cells clamped at various holding potentials were determined. All of the analogues...

  14. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  15. Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors

    Rubio, Fernando; Kamp, Lisa; Carpino, Justin; Faltin, Erin; Loftin, Keith A.; Molgó, Jordi; Aráoz, Rómulo

    2014-01-01

    Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated α-bungarotoxin (the tracer) and streptavidin-horseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts.

  16. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    McCormick, D.J.; Griesmann, G.E.; Huang, Z.; Lennon, V.A.

    1986-03-05

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

  17. Phosphocholine - an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors.

    Richter, K; Mathes, V; Fronius, M; Althaus, M; Hecker, A; Krasteva-Christ, G; Padberg, W; Hone, A J; McIntosh, J M; Zakrzewicz, A; Grau, V

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  18. Phosphocholine – an agonist of metabotropic but not of ionotropic functions of α9-containing nicotinic acetylcholine receptors

    Richter, K.; Mathes, V.; Fronius, M.; Althaus, M.; Hecker, A.; Krasteva-Christ, G.; Padberg, W.; Hone, A. J.; McIntosh, J. M.; Zakrzewicz, A.; Grau, V.

    2016-01-01

    We demonstrated previously that phosphocholine and phosphocholine-modified macromolecules efficiently inhibit ATP-dependent release of interleukin-1β from human and murine monocytes by a mechanism involving nicotinic acetylcholine receptors (nAChR). Interleukin-1β is a potent pro-inflammatory cytokine of innate immunity that plays pivotal roles in host defence. Control of interleukin-1β release is vital as excessively high systemic levels cause life threatening inflammatory diseases. In spite of its structural similarity to acetylcholine, there are no other reports on interactions of phosphocholine with nAChR. In this study, we demonstrate that phosphocholine inhibits ion-channel function of ATP receptor P2X7 in monocytic cells via nAChR containing α9 and α10 subunits. In stark contrast to choline, phosphocholine does not evoke ion current responses in Xenopus laevis oocytes, which heterologously express functional homomeric nAChR composed of α9 subunits or heteromeric receptors containing α9 and α10 subunits. Preincubation of these oocytes with phosphocholine, however, attenuated choline-induced ion current changes, suggesting that phosphocholine may act as a silent agonist. We conclude that phophocholine activates immuno-modulatory nAChR expressed by monocytes but does not stimulate canonical ionotropic receptor functions. PMID:27349288

  19. Inhibitory mechanisms and binding site location for serotonin selective reuptake inhibitors on nicotinic acetylcholine receptors.

    Arias, Hugo R; Feuerbach, Dominik; Bhumireddy, Pankaj; Ortells, Marcelo O

    2010-05-01

    Functional and structural approaches were used to examine the inhibitory mechanisms and binding site location for fluoxetine and paroxetine, two serotonin selective reuptake inhibitors, on nicotinic acetylcholine receptors (AChRs) in different conformational states. The results establish that: (a) fluoxetine and paroxetine inhibit h alpha1beta1 gammadelta AChR-induced Ca(2+) influx with higher potencies than dizocilpine. The potency of fluoxetine is increased approximately 10-fold after longer pre-incubation periods, which is in agreement with the enhancement of [(3)H]cytisine binding to resting but activatable Torpedo AChRs elicited by these antidepressants, (b) fluoxetine and paroxetine inhibit the binding of the phencyclidine analog piperidyl-3,4-(3)H(N)]-(N-(1-(2 thienyl)cyclohexyl)-3,4-piperidine to the desensitized Torpedo AChR with higher affinities compared to the resting AChR, and (c) fluoxetine inhibits [(3)H]dizocilpine binding to the desensitized AChR, suggesting a mutually exclusive interaction. This is supported by our molecular docking results where neutral dizocilpine and fluoxetine and the conformer of protonated fluoxetine with the highest LUDI score interact with the domain between the valine (position 13') and leucine (position 9') rings. Molecular mechanics calculations also evidence electrostatic interactions of protonated fluoxetine at positions 20', 21', and 24'. Protonated dizocilpine bridges these two binding domains by interacting with the valine and outer (position 20') rings. The high proportion of protonated fluoxetine and dizocilpine calculated at physiological pH suggests that the protonated drugs can be attracted to the channel mouth before binding deeper within the AChR ion channel between the leucine and valine rings, a domain shared with phencyclidine, finally blocking ion flux and inducing AChR desensitization. PMID:20079457

  20. Novel positive allosteric modulators of the human α7 nicotinic acetylcholine receptor.

    Arias, Hugo R; Gu, Ruo-Xu; Feuerbach, Dominik; Guo, Bao-Bao; Ye, Yong; Wei, Dong-Qing

    2011-06-14

    The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important. PMID:21510634

  1. Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

    Arias, Hugo R.; Rosenberg, Avraham; Targowska-Duda, Katarzyna M.; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W.

    2015-01-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca2+ influx in hα3β4 AChRs with ~9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (Kd = 0.46 ± 0.06 µM), and ibogaine inhibits [3H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time. PMID:20684041

  2. Functional and structural interaction of (-)-reboxetine with the human α4β2 nicotinic acetylcholine receptor.

    Arias, Hugo R; Fedorov, Nikolai B; Benson, Lisa C; Lippiello, Patrick M; Gatto, Greg J; Feuerbach, Dominik; Ortells, Marcelo O

    2013-01-01

    The interaction of the selective norepinephrine reuptake inhibitor (-)-reboxetine with the human α4β2 nicotinic acetylcholine receptor (nAChR) in different conformational states was studied by several functional and structural approaches. Patch-clamp and Ca(2+)-influx results indicate that (-)-reboxetine does not activate hα4β2 nAChRs via interaction with the orthosteric sites, but inhibits agonist-induced hα4β2 activation by a noncompetitive mechanism. Consistently, the results from the electrophysiology-based functional approach suggest that (-)-reboxetine may act via open channel block; therefore, it is capable of producing a use-dependent type of inhibition of the hα4β2 nAChR function. We tested whether (-)-reboxetine binds to the luminal [(3)H]imipramine site. The results indicate that, although (-)-reboxetine binds with low affinity to this site, it discriminates between the resting and desensitized hα4β2 nAChR ion channels. Patch-clamp results also indicate that (-)-reboxetine progressively inhibits the hα4β2 nAChR with two-fold higher potency at the end of one-second application of agonist, compared with the peak current. The molecular docking studies show that (-)-reboxetine blocks the ion channel at the level of the imipramine locus, between M2 rings 6' and 14'. In addition, we found a (-)-reboxetine conformer that docks in the helix bundle of the α4 subunit, near the middle region. According to molecular dynamics simulations, (-)-reboxetine binding is stable for both sites, albeit less stable than imipramine. The interaction of these drugs with the helix bundle might alter allostericaly the functionality of the channel. In conclusion, the clinical action of (-)-reboxetine may be produced (at least partially) by its inhibitory action on hα4β2 nAChRs. PMID:23010362

  3. Interaction of ibogaine with human alpha3beta4-nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Rosenberg, Avraham; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Yuan, Xiao Juan; Jozwiak, Krzysztof; Moaddel, Ruin; Wainer, Irving W

    2010-09-01

    The interaction of ibogaine and phencyclidine (PCP) with human (h) alpha3beta4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (+/-)-epibatidine-induced Ca2+ influx in h(alpha)3beta4 AChRs with approximately 9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the h(alpha)3beta4 AChR ion channel with relatively high affinity (Kd = 0.46 +/- 0.06 microM), and ibogaine inhibits [3H]ibogaine binding to the desensitized h(alpha)3beta4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the h(alpha)3beta4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6') and valine/phenylalanine (position 13') rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time. PMID:20684041

  4. Structure-activity relationship of ibogaine analogs interacting with nicotinic acetylcholine receptors in different conformational states.

    Arias, Hugo R; Feuerbach, Dominik; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof

    2011-09-01

    The interaction of ibogaine analogs with nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that ibogaine analogs: (a) inhibit (±)-epibatidine-induced Ca²⁺ influx in human embryonic muscle AChRs with the following potency sequence (IC(50) in μM): (±)-18-methylaminocoronaridine (5.9±0.3)∼(±)-18-methoxycoronaridine (18-MC) (6.8±0.8)>(-)-ibogaine (17±3)∼(+)-catharanthine (20±1)>(±)-albifloranine (46±13), (b) bind to the [³H]TCP binding site with higher affinity when the Torpedo AChR is in the desensitized state compared to that in the resting state. Similar results were obtained using [³H]18-MC. These and docking results suggest a steric interaction between TCP and ibogaine analogs for the same site, (c) enhance [³H]cytisine binding to resting but not to desensitized AChRs, with desensitizing potencies (apparent EC₅₀) that correlate very well with the pK(i) values in the desensitized state, and (d) there are good bilinear correlations between the ligand molecular volumes and their affinities in the desensitized and resting states, with an optimal volume of ∼345 ų for the ibogaine site. These results indicate that the size of the binding sites for ibogaine analogs, located between the serine and nonpolar rings and shared with TCP, is an important structural feature for binding and for inducing desensitization. PMID:21642011

  5. Alpha7 nicotinic acetylcholine receptor agonists and PAMs as adjunctive treatment in schizophrenia. An experimental study.

    Marcus, Monica M; Björkholm, Carl; Malmerfelt, Anna; Möller, Annie; Påhlsson, Ninni; Konradsson-Geuken, Åsa; Feltmann, Kristin; Jardemark, Kent; Schilström, Björn; Svensson, Torgny H

    2016-09-01

    Nicotine has been found to improve cognition and reduce negative symptoms in schizophrenia and a genetic and pathophysiological link between the α7 nicotinic acetylcholine receptors (nAChRs) and schizophrenia has been demonstrated. Therefore, there has been a large interest in developing drugs affecting the α7 nAChRs for schizophrenia. In the present study we investigated, in rats, the effects of a selective α7 agonist (PNU282987) and a α7 positive allosteric modulator (PAM; NS1738) alone and in combination with the atypical antipsychotic drug risperidone for their utility as adjunct treatment in schizophrenia. Moreover we also investigated their utility as adjunct treatment in depression in combination with the SSRI citalopram. We found that NS1738 and to some extent also PNU282987, potentiated a subeffective dose of risperidone in the conditioned avoidance response test. Both drugs also potentiated the effect of a sub-effective concentration of risperidone on NMDA-induced currents in pyramidal cells of the medial prefrontal cortex. Moreover, NS1738 and PNU282987 enhanced recognition memory in the novel object recognition test, when given separately. Both drugs also potentiated accumbal but not prefrontal risperidone-induced dopamine release. Finally, PNU282987 reduced immobility in the forced swim test, indicating an antidepressant-like effect. Taken together, our data support the utility of drugs targeting the α7 nAChRs, perhaps especially α7 PAMs, to potentiate the effect of atypical antipsychotic drugs. Moreover, our data suggest that α7 agonists and PAMs can be used to ameliorate cognitive symptoms in schizophrenia and depression. PMID:27474687

  6. [Treatment approach to congenital myasthenic syndrome in a patient with acetylcholine receptor deficiency].

    Ishigaki, Keiko; Murakami, Terumi; Ito, Yasushi; Yanagisawa, Akiko; Kodaira, Kayano; Shishikura, Keiko; Suzuki, Haruko; Hirayama, Yoshito; Osawa, Makiko

    2009-01-01

    Congenital myasthenic syndromes (CMS) are rare heterogeneous disorders of neurotransmission caused by genetic defects of neuromuscular junction molecules. While CMS patients have been reported worldwide, in Japan there have been only a few descriptions of adult CMS patients with acetylcholinesterase (AChE) deficiency and slow channel syndrome. Herein, we report a Japanese CMS patient with acetylcholine receptor (AChR) deficiency, diagnosed during childhood, and our treatment approach to the patient. This 13-year-old Japanese boy had had severe myasthenic symptoms since infancy. Ptosis, his first symptom, appeared at 5 months and nasal voice was recognized at 2 years of age. AchR and anti-muscle-specific tyrosine kinase (Musk) antibody remained negative. A positive tensilon test and decremental response on electromyogram supported the diagnosis of sero-negative myasthenia gravis. Despite thymectomy and strong immunosuppressive therapy including steroid pulse and FK 506, he gradually deteriorated and became wheelchair bound. Genetic analyses for AchR, Rapsyn, Musk and AChE were negative. At age 11 years, a muscle biopsy was performed in the deltoid muscle for neuromuscular junction sampling. Electron microscopic and confocal microscopic analysis of endplates showed almost complete loss of AChR and the diagnosis of CMS with AChR deficiency was confirmed. All immunosuppressive therapies were discontinued. Instead, we started Ubretide and 3,4-diaminopyridine (DAP) after obtaining informed consent. Although not approved in Japan for this use, 3,4-DAP is reportedly effective in refractory cases of CMS. The patient experienced no side effects. Despite all of the objective data were improving, his subjective symptoms and ADL remained poor. There are still many challenges in the treatment of the patient. PMID:19172815

  7. Association of nicotinic acetylcholine receptor subunit alpha-4 polymorphisms with smoking behaviors in Chinese male smokers

    CHU Cheng-jing; YANG Yan-chun; WEI Jin-xue; ZHANG Lan

    2011-01-01

    Background It has been reported that the nicotinic acetylcholine receptor subunit a4 gene (CHRNA4) might be associated with smoking behaviors in the previous studies. Up to now, there are few reports on the relationship between CHRNA4 and smoking initiation. In this study, we tried to explore the role of two polymorphisms in CHRNA4 (rs 1044396 and rs 1044397) in smoking initiation and nicotine dependence in Chinese male smokers.Methods Nine hundred and sixty-six Chinese male lifetime nonsmokers and smokers were assessed by the Fagerstr(o)m test for nicotine dependence (FTND), smoking quantity (SQ) and the heaviness of smoking index (HSI). All subjects were divided into four groups based on their tobacco use history and the FTND scores. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to find two polymorphisms of CHRNA4 in these subjects.Results The x2 test showed that rs1044396 was significantly associated with smoking initiation (x2=4.65, P=0.031),while both rs1044396 and rs1044397 were significantly associated with nicotine dependence (x2=5.42, P=0.020; x2=758,P=0.005). Furthermore, the T-G (3.9%) haplotype of rs1044396-rs1044397 showed significant association with smoking initiation (x2=6.30, P=0.012) and the C-G haplotype (58.9%) remained positive association with nicotine dependence (x2=8.64, P=0.003) after Bonferroni correction. The C-G haplotype also significantly increased the HSI (P=0.002) and FTND scores (P=0.001) after Bonferroni correction.Conclusion These findings suggest that CHRNA4 may be associated with smoking initiation and the C-G haplotype of rs1044396-rs1044397 might increase the vulnerability to nicotine dependence in Chinese male smokers.

  8. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of [3H]-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. [3H]-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties

  9. Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: Pharmacological and biochemical properties

    Buck, M.A.; Fraser, C.M. (National Institute on Alcohol Abuse and Alcoholism, Rockville, MD (USA))

    1990-12-14

    The pharmacological and biochemical properties of rat m1 and m3 muscarinic acetylcholine receptors (mAChR) stably transfected into Chinese hamster ovary-K1 (CHO) cells were characterized with ligand binding, affinity labeling and biochemical assays. Both mAChR subtypes display saturable, high affinity binding of (3H)-quinuclidinyl benzilate (QNB) and a rank order of antagonist potency of QNB greater than atropine greater than pirenzepine greater than AF-DX 116. Carbachol displacement of (3H)-QNB binding to the m3 mAChR revealed an approximate 17-fold higher affinity than observed with the m1 mAChR. (3H)-propylbenzilylcholine mustard (PrBCM) labeling of mAChR revealed that m1 and m3 mAChR migrated on SDS-polyacrylamide gels with apparent molecular masses of 80,000 and 94,000 daltons, respectively, consistent with the known differences in their molecular sizes. Both m1 and m3 mAChR elicited dose-dependent increases in the hydrolysis of phosphoinositides; however, the maximal increase in total inositol phosphates elicited with the m1 mAChR was approximately 2-fold greater than that observed in cells expressing similar densities of m3 mAChR. Agonist activation of the m1 mAChR also elicited increases in basal and forskolin-stimulated cAMP, whereas the m3 mAChR had no effect on intracellular cAMP levels. These data suggest that although m1 and m3 mAChR display a considerable degree of structural homology, they exhibit distinct pharmacological and biochemical properties.

  10. alpha4beta2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief

    McGranahan, Tresa M.; Patzlaff, Natalie E.; Grady, Sharon R.; Heinemann, Stephen F.; Booker, T.K.

    2012-01-01

    Nicotine is the primary psychoactive substance in tobacco and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the alpha4beta2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal, as well as nicotine-induced, behaviors. Although alpha4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addition, mental illness and movement control in humans. We developed a unique model system to examine the role of alpha4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the alpha4 subunit from dopaminergic neurons in mice. The loss alpha4 mRNA and alpha4beta2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of alpha4beta2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. Alpha4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. Alpha4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze and elimination of alpha4-beta2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of alpha4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression, however nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine behaviors. PMID:21795541

  11. α4β2 nicotinic acetylcholine receptors on dopaminergic neurons mediate nicotine reward and anxiety relief.

    McGranahan, Tresa M; Patzlaff, Natalie E; Grady, Sharon R; Heinemann, Stephen F; Booker, T K

    2011-07-27

    Nicotine is the primary psychoactive substance in tobacco, and it exerts its effects by interaction with various subtypes of nicotinic acetylcholine receptors (nAChRs) in the brain. One of the major subtypes expressed in brain, the α4β2-nAChR, endogenously modulates neuronal excitability and thereby, modifies certain normal as well as nicotine-induced behaviors. Although α4-containing nAChRs are widely expressed across the brain, a major focus has been on their roles within midbrain dopaminergic regions involved in drug addiction, mental illness, and movement control in humans. We developed a unique model system to examine the role of α4-nAChRs within dopaminergic neurons by a targeted genetic deletion of the α4 subunit from dopaminergic neurons in mice. The loss α4 mRNA and α4β2-nAChRs from dopaminergic neurons was confirmed, as well as selective loss of α4β2-nAChR function from dopaminergic but not GABAergic neurons. Two behaviors central to nicotine dependence, reward and anxiety relief, were examined. α4-nAChRs specifically on dopaminergic neurons were demonstrated to be necessary for nicotine reward as measured by nicotine place preference, but not for another drug of addiction, cocaine. α4-nAChRs are necessary for the anxiolytic effects of nicotine in the elevated plus maze, and elimination of α4β2-nAChRs specifically from dopaminergic neurons decreased sensitivity to the anxiolytic effects of nicotine. Deletion of α4-nAChRs specifically from dopaminergic neurons also increased sensitivity to nicotine-induced locomotor depression; however, nicotine-induced hypothermia was unaffected. This is the first work to develop a dopaminergic specific deletion of a nAChR subunit and examine resulting changes in nicotine-related behaviors. PMID:21795541

  12. Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

    Qi-Guang Shi; Zhi-Hong Wang; Xiao-Wei Ma; Da-Qi Zhang; Chun-Sheng Yang; Fu-Dong Shi; Li Yang

    2012-01-01

    Objective To evaluate the frequency,distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG).Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n =90) and generalized MG (GMG) patients (n =110).The fetaltype (2α∶ β∶ γ∶ δ) and adult-type (2α∶ β∶ ε∶ δ) AChR were used as antigens,and their relevance to disease presentation was assessed.Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined,with 14 having serum specific to the AChR-γ subunit,and 22 to the AChR-ε subunit.The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P =0.015).Compared with OMG patients,the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG.Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR),1.03;95% confidence interval (CI),1.01-1.06 and OR,5.09;95% CI,2.23-11.62].Conclusion Using both fetal-and adulttype AChRs as the antigens may be more sensitive than using either subtype.Patients with serum specific to both isoforms are at a greater risk of progressing to GMG.Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.

  13. Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

    Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

    2015-11-01

    The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity. PMID:25998908

  14. Chronic nicotine modifies skeletal muscle Na,K-ATPase activity through its interaction with the nicotinic acetylcholine receptor and phospholemman.

    Alexander V Chibalin

    Full Text Available Our previous finding that the muscle nicotinic acetylcholine receptor (nAChR and the Na,K-ATPase interact as a regulatory complex to modulate Na,K-ATPase activity suggested that chronic, circulating nicotine may alter this interaction, with long-term changes in the membrane potential. To test this hypothesis, we chronically exposed rats to nicotine delivered orally for 21-31 days. Chronic nicotine produced a steady membrane depolarization of ∼3 mV in the diaphragm muscle, which resulted from a net change in electrogenic transport by the Na,K-ATPase α2 and α1 isoforms. Electrogenic transport by the α2 isoform increased (+1.8 mV while the activity of the α1 isoform decreased (-4.4 mV. Protein expression of Na,K-ATPase α1 or α2 isoforms and the nAChR did not change; however, the content of α2 subunit in the plasma membrane decreased by 25%, indicating that its stimulated electrogenic transport is due to an increase in specific activity. The physical association between the nAChR, the Na,K-ATPase α1 or α2 subunits, and the regulatory subunit of the Na,K-ATPase, phospholemman (PLM, measured by co-immuno precipitation, was stable and unchanged. Chronic nicotine treatment activated PKCα/β2 and PKCδ and was accompanied by parallel increases in PLM phosphorylation at Ser(63 and Ser(68. Collectively, these results demonstrate that nicotine at chronic doses, acting through the nAChR-Na,K-ATPase complex, is able to modulate Na,K-ATPase activity in an isoform-specific manner and that the regulatory range includes both stimulation and inhibition of enzyme activity. Cholinergic modulation of Na,K-ATPase activity is achieved, in part, through activation of PKC and phosphorylation of PLM.

  15. “Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

    Huang, Hai-peng; Pan, Hong; Wang, Hong-feng

    2016-01-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. “Warming yang and invigorating qi” acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following “warming yang and invigorating qi” acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that “warming yang and invigorating qi” acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  16. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis.

    Huang, Hai-Peng; Pan, Hong; Wang, Hong-Feng

    2016-03-01

    Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10), Zusanli (ST36), Pishu (BL20), and Shenshu (BL23) once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis. PMID:27127487

  17. "Warming yang and invigorating qi" acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

    Hai-peng Huang

    2016-01-01

    Full Text Available Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction. "Warming yang and invigorating qi" acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis. However, few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction. Here, we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following "warming yang and invigorating qi" acupuncture therapy. Needles were inserted at acupressure points Shousanli (LI10, Zusanli (ST36, Pishu (BL20, and Shenshu (BL23 once daily for 7 consecutive days. The treatment was repeated after 1 day of rest. We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment. This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide. These findings suggest that "warming yang and invigorating qi" acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.

  18. The α4β2 nicotine acetylcholine receptor agonist ispronicline induces c-Fos expression in selective regions of the rat forebrain

    Jacobsen, Julie; Hansen, Henrik H; Kiss, Alexander;

    2012-01-01

    The dominant nicotine acetylcholine receptor (nAChR) subtype in the brain is the pentameric receptor containing both α4 and β2 subunits (α4β2). Due to the lack of selective agonists it has not been ruled out what neuronal circuits that are stimulated after systemic administration with nicotine. We...

  19. The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori

    Zhang Chuan-Xi

    2007-09-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. Results We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR and rapid amplification of cDNA ends (RACE methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine α-type subunits and three β-type subunits. The silkworm possesses three genes having low identity with others, including one α and two β subunits, α9, β2 and β3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene α6 has RNA-editing sites, and α4, α6 and α8 undergo alternative splicing. In particular, alternative exon 7 of Bmα8 may have arisen from a recent duplication event. Truncated transcripts were found for Bmα4 and Bmα5. Conclusion B. mori possesses a largest known insect nAChR gene family characterized to date, including nine α-type subunits and three β-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family.

  20. Neonicotinoid binding, toxicity and expression of nicotinic acetylcholine receptor subunits in the aphid Acyrthosiphon pisum.

    Emiliane Taillebois

    Full Text Available Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI, thiamethoxam (TMX and clothianidin (CLT. Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16 ± 0.04 nM and 41.7 ± 5.9 nM and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008 ± 0.002 nM and 1.135 ± 0.213 nM. Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml and TMX (LC50 = 0.034 µg/ml were more toxic than CLT (LC50 = 0.118 µg/ml. The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.

  1. Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes.

    Wenz, Jorge J; Barrantes, Francisco J

    2005-01-11

    Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup

  2. Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

    Fernández-Carvajal, Asia M; Encinar, José A; Poveda, José Antonio; de Juan, Entilio; Martínez-Pinna, Juan; Ivorra, Isabel; Ferragut, José Antonio; Morales, Andrés; González-Ros, José Manuel

    2006-01-01

    Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of

  3. Synthesis and evaluation of new imaging agent for central nicotinic acetylcholine receptor α7 subtype

    Introduction: The nicotinic acetylcholine receptor (nAChR) α7 subtype (α7 nAChR) is one of the major nAChR subtypes in the brain. We synthesized C-11 labeled α7 nAChR ligands, (R)-2-[11C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([11C](R)-MeQAA) and its isomer (S)-[11C]MeQAA, for in vivo investigation with positron emission tomography (PET). Then, the potential of (R)- and (S)-[11C]MeQAA for in vivo imaging of α7 nAChR in the brain was evaluated in mice and monkeys. Methods: The binding affinity for α7 nAChR was measured using rat brain. Biodistribution and in vivo receptor blocking studies were undertaken in mice. Dynamic PET scans were performed in conscious monkeys. Results: The affinity for α7 nAChR was 41 and 182 nM for (R)- and (S)-MeQAA, respectively. The initial uptake in the mouse brain was high ([11C](R)-MeQAA: 7.68 and [11C](S)-MeQAA: 6.65 %dose/g at 5 min). The clearance of [11C](R)-MeQAA was slow in the hippocampus (α7 nAChR-rich region) but was rapid in the cerebellum (α7 nAChR-poor region). On the other hand, the clearance was fast for [11C](S)-MeQAA in all regions. The brain uptake of [11C](R)-MeQAA was decreased by methyllycaconitine (α7 nAChR antagonist) treatment. In monkeys, α7 nAChRs were highly distributed in the thalamus and cortex but poorly distributed in the cerebellum. The high accumulation was observed in the cortex and thalamus for [11C](R)-MeQAA, while the uptake was rather homogeneous for [11C](S)-MeQAA. Conclusions: [11C](R)-MeQAA was successfully synthesized and showed high uptake to the brain. However, since the in vivo selectivity for α7 nAChR was not enough, further PET kinetic analysis or structure optimization is needed for specific visualization of brain α7 nAChRs in vivo.

  4. Effect of tissue-specific acetylcholinesterase inhibitor C-547 on α3β4 and αβεδ acetylcholine receptors in COS cells.

    Lindovský, Jiří; Petrov, Konstantin; Krůšek, Jan; Reznik, Vladimir S; Nikolsky, Eugeny E; Vyskočil, František

    2012-08-01

    The C-547 is the most effective muscle and tissue-specific anticholinesterase among alkylammonium derivatives of 6-methyluracil (ADEMS) acting in nanomolar concentrations on locomotor muscles but not on respiratory muscles, smooth muscles and heart and brain acetylcholine esterases (AChE). When applied systematically it could influence peripheral acetylcholine receptors. The aim of the present study was to investigate the effect of C-547 on rat α3β4 (ganglionic type) and αβεδ (muscle type) nicotinic receptors expressed in COS cells. Currents evoked by rapid application of acetylcholine or nicotine were recorded in whole-cell mode by electrophysiological patch-clamp technique 2-4 days after cell transfection by plasmids coding the α3β4 or αβεδ combination of receptor subunits. In cells sensitive to acetylcholine, the application of C-547 evoked no responses. When acetylcholine was applied during an already running application of C-547, acetylcholine responses were only inhibited at concentrations higher than 10(-7)M. This inhibition is not voltage-dependent, but is accompanied by an increased rate of desensitization. Thus in both types of receptors, effective doses are approximately 100 times higher than those inhibiting AChE in leg muscles and similar to those inhibiting respiratory diaphragm muscles and external intercostal muscles. These observations show that C-547 can be considered for symptomatic treatment of myasthenia gravis and other congenital myasthenic syndromes as an inhibitor of AChE in leg muscles at concentrations much lower than those inhibiting muscle and ganglion types of acetylcholine receptors. PMID:22634638

  5. Oseltamivir produces hypothermic and neuromuscular effects by inhibition of nicotinic acetylcholine receptor functions: comparison to procaine and bupropion.

    Fukushima, Akihiro; Chazono, Kaori; Hashimoto, Yuichi; Iwajima, Yui; Yamamoto, Shohei; Maeda, Yasuhiro; Ohsawa, Masahiro; Ono, Hideki

    2015-09-01

    Oseltamivir, an anti-influenza virus drug, induces marked hypothermia in normal mice. We have proposed that the hypothermic effect arises from inhibition of the nicotinic acetylcholine receptor function of sympathetic ganglion neurons which innervate the brown adipose tissue (a heat generator). It has been reported that local anesthetics inhibit nicotinic acetylcholine receptor function by acting on its ionic channels, and that bupropion, a nicotinic antagonist, induces hypothermia. In this study, we compared the effects of oseltamivir, procaine and bupropion on body temperature, cardiovascular function and neuromuscular transmission. Intraperitoneal administration of oseltamivir (100mg/kg), procaine (86.6mg/kg) and bupropion (86.7mg/kg) lowered the core body temperature of normal mice. At lower doses (10-30mg/kg oseltamivir, 8.7-26mg/kg procaine and bupropion), when administered subcutaneously, the three drugs antagonized the hypothermia induced by intraperitoneal injection of nicotine (1mg/kg). In anesthetized rats, intravenous oseltamivir (30-100mg/kg), procaine (10mg/kg) and bupropion (10mg/kg) induced hypotension and bradycardia. Oseltamivir alone (100mg/kg) did not inhibit neuromuscular twitch contraction of rats, but at 3-30mg/kg it augmented the muscle-relaxing effect of d-tubocurarine. Similar effects were observed when lower doses of procaine (10-30mg/kg) and bupropion (3-10mg/kg) were administered, suggesting that systemic administration of oseltamivir inhibits muscular nicotinic acetylcholine receptors. These results support the idea that the hypothermic effect of oseltamivir is due to its effects on sympathetic ganglia which innervate the brown adipose tissue, and suggest that oseltamivir may exert non-selective ion channel blocking effects like those of ester-type local anesthetics. PMID:26049014

  6. Calcium-dependent effect of the thymic polypeptide thymopoietin on the desensitization of the nicotinic acetylcholine receptor

    The effects of the thymic polypeptide thymopoietin (Tpo) on the properties of the nicotinic acetylcholine receptor (AcChoR) were investigated by patch clamp techniques on mouse C2 myotubes and by biochemical assays on AcChoR-rich membrane fragments purified from the Torpedo marmorata electric organ. At high concentrations (> 100 nM), Tpo inhibits the binding of cholinergic agonists to the AcChoR in a Ca2+-insensitive manner. At lower concentrations (2 nM), Tpo applied on C2 myotubes simultaneously with nondesensitizing concentrations of acetylcholine results in the appearance of long closed times separating groups of openings. This effect depends on the presence of Ca2+ in the external medium. Outside-out recordings, performed with various concentrations of EGTA in the intracellular medium, suggest that Ca2+ acts on the cytoplasmic face of the membrane after entry through acetylcholine-activated channels. Parallel studies with T. marmorata AcChoR-rich membranes show that in the presence of Ca2+ Tpo causes a decrease in the apparent equilibrium dissociation constant of the noncompetitive blocker [3H]phencyclidine, enhances, at low concentrations, the binding of [3H]acetylcholine, and also alters the binding kinetics of the fluorescent agonist 6-(5-dimethylamino-1-naphthalenesulfonamido)-n-hexanoic acid β-(N-trimethylammonium bromide) ethyl ester to the AcChoR. It was concluded that, in the presence of Ca2+, Tpo displaces the conformational equilibrium of the AcChoR towards a high-affinity desensitized state and increases the transition rate towards the same state

  7. Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M2 and M4 muscarinic receptors

    Machová, Eva; Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

    Praha : 2.Lékařská fakulta UK, 2007. s. 55-55. [Vědecká konference 2007 - věda, sport a rock ´n´roll. 25.04.2007-26.04.2007, Praha] R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant ostatní: NIH(US) NS25732 Institutional research plan: CEZ:AV0Z50110509 Keywords : spo2 * xanomeline * acetylcholine * presynaptic muscarinic receptors Subject RIV: FH - Neurology

  8. Acetylcholine sensitivity of biphasic Ca2+ mobilization induced by nicotinic receptor activation at the mouse skeletal muscle endplate

    Dezaki, Katsuya; Kimura, Ikuko

    1998-01-01

    Acetylcholine (ACh) was locally applied onto the endplate region in a mouse phrenic nerve-diaphragm muscle preparation to measure intracellular free calcium ([Ca2+]i) entry through nicotinic ACh receptors (AChRs) by use of Ca2+-aequorin luminescence.ACh (0.1–3 mM, 20 μl) elicited biphasic elevation of [Ca2+]i (fast and slow Ca2+ mobilization) in muscle cells. The peak amplitude of the slow Ca2+ mobilization (not accompanied by twitch tension) was concentration-dependently increased by ACh, wh...

  9. Expression of the α-bungarotoxin binding site of the nicotinic acetylcholine receptor by Escherichia coli transformants

    Restriction fragments of DNA derived from a cDNA clone of the α subunit of the acetylcholine receptor were subcloned in Escherichia coli by using the trpE fusion vector, pATH2. Transformants expressing the amino acid sequences 166-315 or 166-200 are shown to produce a chimeric protein that bound α-bungarotoxin. Moreover, it is shown that sufficient amounts of toxin-binding proteins can be generated by individual colonies of bacteria. This provides a new approach for gene selection via functional expression-i.e., ligand overlays of colony blots

  10. The regulation of M1 muscarinic acetylcholine receptor desensitization by synaptic activity in cultured hippocampal neurons1

    Willets, Jonathon M.; Nelson, Carl P.; Nahorski, Stefan R; Challiss, R.A. John

    2007-01-01

    To better understand metabotropic/ionotropic integration in neurons we have examined the regulation of M1 muscarinic acetylcholine (mACh) receptor signalling in mature (> 14 days in vitro), synaptically-active hippocampal neurons in culture. Using a protocol where neurons are exposed to an EC50 concentration of the muscarinic agonist methacholine (MCh) prior to (R1), and following (R2) a desensitizing pulse of a high concentration of this agonist, we have found that the reduction in M1 mACh r...

  11. Structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by cross-reacting monoclonal antibodies

    1984-01-01

    A collection of 126 monoclonal antibodies (mAbs) made against acetylcholine receptors (AChRs) from the electric organs of Torpedo californica or Electrophorus electricus was tested for cross-reactivity with AChRs in cryostat sections of skeletal muscle from Rana pipiens and Xenopus laevis by indirect immunofluorescence. 49 mAbs (39%) cross- reacted with AChRs from Rana, and 25 mAbs (20%) cross-reacted with AChRs from Xenopus. mAbs specific for each of the four subunits of electric organ AChR ...

  12. Carbamoylcholine analogs as nicotinic acetylcholine receptor agonists--structural modifications of 3-(dimethylamino)butyl dimethylcarbamate (DMABC)

    Hansen, Camilla Petrycer; Jensen, Anders Asbjørn; Balle, Thomas;

    2009-01-01

    Compounds based on the 3-(dimethylamino)butyl dimethylcarbamate (DMABC) scaffold were synthesized and pharmacologically characterized at the alpha(4)beta(2), alpha(3)beta(4,) alpha(4)beta(4) and alpha(7) neuronal nicotinic acetylcholine receptors (nAChRs). The carbamate functionality and a small...... hydrophobic substituent in the C-3 position were found to be vital for the binding affinity to the nAChRs, whereas the carbamate nitrogen substituents were important for nAChR subtype selectivity. Finally, the compounds were found to be agonists at the alpha(3)beta(4) nAChR....

  13. Bupropion-induced inhibition of α7 nicotinic acetylcholine receptors expressed in heterologous cells and neurons from dorsal raphe nucleus and hippocampus.

    Vázquez-Gómez, Elizabeth; Arias, Hugo R; Feuerbach, Dominik; Miranda-Morales, Marcela; Mihailescu, Stefan; Targowska-Duda, Katarzyna M; Jozwiak, Krzysztof; García-Colunga, Jesús

    2014-10-01

    The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation. PMID:25016090

  14. Effect of α7 nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    Nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChRs located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The toxicity and teratogenicity of many plants (which results in millions of dollars in losses annually to the livestock industry) are due to various toxins that bind to nAChRs including deltaline and methyllycaconitine (MLA) from larkspur (Delphinium) species, and nicotine and anabasine from tobacco (Nicotiana) species. The primary result of the actions of these alkaloids at nAChRs is neuromuscular paralysis and respiratory failure. The objective of this study was to further characterize the motor coordination deficiencies that occur upon exposure to a non-lethal dose of nAChR antagonists MLA and deltaline as well as nAChR agonists nicotine and anabasine. We evaluated the effect of nAChR agonists and antagonists on the motor function and coordination in mice using a balance beam, grip strength meter, rotarod, open field analysis and tremor monitor. These analyses demonstrated that within seconds after treatment the mice had significant loss of motor function and coordination that lasted up to 1 min, followed by a short period of quiescence. Recovery to normal muscle coordination was rapid, typically within approximately 10 min post-dosing. However, mice treated with the nAChR agonist nicotine and anabasine required a slightly longer time to recover some aspects of normal muscle function in comparison to mice treated with the nAChR antagonist MLA or deltaline. -- Highlights: ► Mice treated with nAChR agonists and antagonists have a loss in motor function. ► These deficits are temporary as near normal motor function returns within 10 min. ► There are compound-specific differences in the effects on motor function.

  15. Functional Characterization of CCHamide and Muscarinic Acetylcholine Receptor Signalling in Drosophila melanogaster

    Ren, Guilin Robin

    mutants created with the CRISP/Cas9 technique showed thatCCHamide-2 is probly an orexigenic peptide and also that is an important factor for larvaldevelopmental timing.In mammals, muscarinic acetylcholine signalling is involved in the signal transmission of theparasympathetic nervous system. However...

  16. Mechanisms of the inhibition of endplate acetylcholine receptors by antiseptic chlorhexidine (experiments and models)

    Shaihutdinova, A.R.; Nikolsky, E. E.; Vyskočil, František; Skorinkin, A.I.

    2009-01-01

    Roč. 380, č. 6 (2009), s. 551-560. ISSN 0028-1298 R&D Projects: GA AV ČR(CZ) IAA500110905 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine * endplate currents Subject RIV: ED - Physiology Impact factor: 2.631, year: 2009

  17. Mood and anxiety regulation by nicotinic acetylcholine receptors: A potential pathway to modulate aggression and related behavioral states.

    Picciotto, Marina R; Lewis, Alan S; van Schalkwyk, Gerrit I; Mineur, Yann S

    2015-09-01

    The co-morbidity between smoking and mood disorders is striking. Preclinical and clinical studies of nicotinic effects on mood, anxiety, aggression, and related behaviors, such as irritability and agitation, suggest that smokers may use the nicotine in tobacco products as an attempt to self-medicate symptoms of affective disorders. The role of nicotinic acetylcholine receptors (nAChRs) in circuits regulating mood and anxiety is beginning to be elucidated in animal models, but the mechanisms underlying the effects of nicotine on aggression-related behavioral states (ARBS) are still not understood. Clinical trials of nicotine or nicotinic medications for neurological and psychiatric disorders have often found effects of nicotinic medications on ARBS, but few trials have studied these outcomes systematically. Similarly, the increase in ARBS resulting from smoking cessation can be resolved by nicotinic agents, but the effects of nicotinic medications on these types of mental states and behaviors in non-smokers are less well understood. Here we review the literature on the role of nAChRs in regulating mood and anxiety, and subsequently on the closely related construct of ARBS. We suggest avenues for future study to identify how nAChRs and nicotinic agents may play a role in these clinically important areas. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25582289

  18. TC-1734: an orally active neuronal nicotinic acetylcholine receptor modulator with antidepressant, neuroprotective and long-lasting cognitive effects.

    Gatto, Gregory J; Bohme, G Andrees; Caldwell, William S; Letchworth, Sharon R; Traina, Vincent M; Obinu, M Carmen; Laville, Michel; Reibaud, Michel; Pradier, Laurent; Dunbar, Geoffrey; Bencherif, Merouane

    2004-01-01

    The development of selective ligands targeting neuronal nicotinic acetylcholine receptors to alleviate symptoms associated with neurodegenerative diseases presents the advantage of affecting multiple deficits that are the hallmarks of these pathologies. TC-1734 is an orally active novel neuronal nicotinic agonist with high selectivity for neuronal nicotinic receptors. Microdialysis studies indicate that TC-1734 enhances the release of acetylcholine from the cortex. TC-1734, by either acute or repeated administration, exhibits memory enhancing properties in rats and mice and is neuroprotective following excitotoxic insult in fetal rat brain in cultures and against alterations of synaptic transmission induced by deprivation of glucose and oxygen in hippocampal slices. At submaximal doses, TC-1734 produced additive cognitive effects when used in combination with tacrine or donepezil. Unlike (-)-nicotine, behavioral sensitization does not develop following repeated administration of TC-1734. Its pharmacokinetic (PK) profile (half-life of 2 h) contrasts with the long lasting improvement in working memory (18 h) demonstrating that cognitive improvement extends beyond the lifetime of the compound. The very low acute toxicity of TC-1734 and its receptor activity profile provides additional mechanistic basis for its suggested potential as a clinical candidate. TC-1734 was very well tolerated in acute and chronic oral toxicity studies in mice, rats and dogs. Phase I clinical trials demonstrated TC-1734's favorable pharmacokinetic and safety profile by acute oral administration at doses ranging from 2 to 320 mg. The bioavailability, pharmacological, pharmacokinetic, and safety profile of TC-1734 provides an example of a safe, potent and efficacious neuronal nicotinic modulator that holds promise for the management of the hallmark symptomatologies observed in dementia. PMID:15179444

  19. Rapid antidepressant actions of scopolamine: Role of medial prefrontal cortex and M1-subtype muscarinic acetylcholine receptors.

    Navarria, Andrea; Wohleb, Eric S; Voleti, Bhavya; Ota, Kristie T; Dutheil, Sophie; Lepack, Ashley E; Dwyer, Jason M; Fuchikami, Manabu; Becker, Astrid; Drago, Filippo; Duman, Ronald S

    2015-10-01

    Clinical studies demonstrate that scopolamine, a non-selective muscarinic acetylcholine receptor (mAchR) antagonist, produces rapid therapeutic effects in depressed patients, and preclinical studies report that the actions of scopolamine require glutamate receptor activation and the mechanistic target of rapamycin complex 1 (mTORC1). The present study extends these findings to determine the role of the medial prefrontal cortex (mPFC) and specific muscarinic acetylcholine receptor (M-AchR) subtypes in the actions of scopolamine. The administration of scopolamine increases the activity marker Fos in the mPFC, including the infralimbic (IL) and prelimbic (PrL) subregions. Microinfusions of scopolamine into either the IL or the PrL produced significant antidepressant responses in the forced swim test, and neuronal silencing of IL or PrL blocked the antidepressant effects of systemic scopolamine. The results also demonstrate that the systemic administration of a selective M1-AChR antagonist, VU0255035, produced an antidepressant response and stimulated mTORC1 signaling in the PFC, similar to the actions of scopolamine. Finally, we used a chronic unpredictable stress model as a more rigorous test of rapid antidepressant actions and found that a single dose of scopolamine or VU0255035 blocked the anhedonic response caused by CUS, an effect that requires the chronic administration of typical antidepressants. Taken together, these findings indicate that mPFC is a critical mediator of the behavioral actions of scopolamine and identify the M1-AChR as a therapeutic target for the development of novel and selective rapid-acting antidepressants. PMID:26102021

  20. Natural Compounds Interacting with Nicotinic Acetylcholine Receptors: From Low-Molecular Weight Ones to Peptides and Proteins

    Denis Kudryavtsev

    2015-05-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs fulfill a variety of functions making identification and analysis of nAChR subtypes a challenging task. Traditional instruments for nAChR research are d-tubocurarine, snake venom protein α-bungarotoxin (α-Bgt, and α-conotoxins, neurotoxic peptides from Conus snails. Various new compounds of different structural classes also interacting with nAChRs have been recently identified. Among the low-molecular weight compounds are alkaloids pibocin, varacin and makaluvamines C and G. 6-Bromohypaphorine from the mollusk Hermissenda crassicornis does not bind to Torpedo nAChR but behaves as an agonist on human α7 nAChR. To get more selective α-conotoxins, computer modeling of their complexes with acetylcholine-binding proteins and distinct nAChRs was used. Several novel three-finger neurotoxins targeting nAChRs were described and α-Bgt inhibition of GABA-A receptors was discovered. Information on the mechanisms of nAChR interactions with the three-finger proteins of the Ly6 family was found. Snake venom phospholipases A2 were recently found to inhibit different nAChR subtypes. Blocking of nAChRs in Lymnaea stagnalis neurons was shown for venom C-type lectin-like proteins, appearing to be the largest molecules capable to interact with the receptor. A huge nAChR molecule sensible to conformational rearrangements accommodates diverse binding sites recognizable by structurally very different compounds.

  1. Solid-phase synthesis and pharmacological evaluation of analogues of PhTX-12-A potent and selective nicotinic acetylcholine receptor antagonist

    Strømgaard, Kristian; Mellor, Ian R; Andersen, Kim;

    2002-01-01

    Philanthotoxin-12 (PhTX-12) is a novel potent and selective, noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs). Homologues of PhTX-12 with 7-11 methylene groups between the primary amino group and the aromatic head-group were synthesized using solid-phase methodology. In vit...

  2. Antidepressant-like effects of nicotinic acetylcholine receptor antagonists, but not agonists, in the mouse forced swim and mouse tail suspension tests

    Andreasen T., Jesper; Olsen, G M; Wiborg, O;

    2009-01-01

    Current literature suggests involvement of nicotinic acetylcholine receptors (nAChRs) in major depression. However, it is controversial whether the antidepressant-like effect of nAChR modulation is induced by activation, desensitization or inhibition of central nAChRs. In addition, the specific n...

  3. Pharmacological characterisation of α6β4* nicotinic acetylcholine receptors assembled from three different α6/α3 subunit chimeras in tsA201 cells

    Jensen, Anne Bjørnskov; Hoestgaard-Jensen, Kirsten; Jensen, Anders A.

    2014-01-01

    on these results should be made keeping the molecular modifications in the α6 surrogate subunits in mind, this study sheds light on the pharmacological properties of α6β4⁎ nicotinic acetylcholine receptors and demonstrates the applicability of the C6F223L and C16F223L chimeras for studies of these...

  4. The α7 nicotinic acetylcholine receptor ligands methyllycaconitine, NS6740 and GTS-21 reduce lipopolysaccharide-induced TNF-α release from microglia

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    The anti-inflammatory properties of, particularly the α7, nicotinic acetylcholine receptors (nAChRs) in the peripheral immune system are well documented. There are also reports of anti-inflammatory actions of nicotine in the CNS, but it is unclear, whether this is due to activation or inhibition ...

  5. Combined α7 nicotinic acetylcholine receptor agonism and partial serotonin transporter inhibition produce antidepressant-like effects in the mouse forced swim and tail suspension tests

    Andreasen, Jesper T; Redrobe, John P; Nielsen, Elsebet Ø

    2012-01-01

    Emerging evidence points to an involvement of nicotinic acetylcholine receptors (nAChRs) in major depression. Nicotine improves symptoms of depression in humans and shows antidepressant-like effects in rodents. Monoamine release is facilitated by nAChR stimulation, and nicotine-evoked serotonin (...

  6. 11C-NS14492 as a novel PET radioligand for imaging cerebral alpha7 nicotinic acetylcholine receptors: in vivo evaluation and drug occupancy measurements

    Ettrup, Anders; Mikkelsen, Jens D; Lehel, Szabolcs; Madsen, Jacob; Nielsen, Elsebet Ø; Palner, Mikael; Timmermann, Daniel B; Peters, Dan; Knudsen, Gitte M

    2011-01-01

    Small-molecule α(7) nicotinic acetylcholine receptor (α(7)nAChR) agonists are currently validated for use as treatment for cognitive disturbances in schizophrenia and in Alzheimer disease. A suitable radiolabeled α(7)nAChR PET tracer would be important for in vivo quantification of α(7)nAChR bind...

  7. A synthetic combinatorial strategy for developing a-conotoxin analogs as potent a7 nicotinic acetylcholine receptor antagonists

    Armishaw, Christopher J; Singh, Narender; Medina-Franco, Jose L; Clark, Richard J; Scott, Krystle C M; Houghten, Richard A; Jensen, Anders Asbjørn

    2010-01-01

    alpha-Conotoxins are peptide neurotoxins isolated from venomous cone snails that display exquisite selectivity for different subtypes of nicotinic acetylcholine receptors (nAChR). They are valuable research tools that have profound implications in the discovery of new drugs for a myriad of...

  8. PASSIVE-AVOIDANCE TRAINING INDUCES ENHANCED LEVELS OF IMMUNOREACTIVITY FOR MUSCARINIC ACETYLCHOLINE-RECEPTOR AND COEXPRESSED PKC-GAMMA AND MAP-2 IN RAT CORTICAL-NEURONS

    VANDERZEE, EA; DOUMA, BRK; BOHUS, B; LUITEN, PGM

    1994-01-01

    Changes in neocortical immunoreactivity (ir) for muscarinic acetylcholine receptors (mAChRs), protein kinase C gamma (PKC gamma), microtubule-associated protein 2 (MAP-2), and the calcium-binding protein parvalbumin (PARV) induced by the performance of a one-trial passive shock avoidance (PSA) task

  9. Structural and functional studies of the modulator NS9283 reveal agonist-like mechanism of action at α4β2 nicotinic acetylcholine receptors

    Olsen, Jeppe A; Ahring, Philip K; Kastrup, Jette Sandholm Jensen;

    2014-01-01

    Modulation of Cys loop receptor ion channels is a proven drug discovery strategy, but many underlying mechanisms of the mode of action are poorly understood. We report the x-ray structure of the acetylcholine-binding protein from Lymnaea stagnalis with NS9283, a stoichiometry selective positive m...

  10. Type I and II positive allosteric modulators differentially modulate agonist-induced up-regulation of α7 nicotinic acetylcholine receptors

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type...