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Sample records for acetylcholine

  1. Acetylcholine receptor antibody

    Science.gov (United States)

    ... medlineplus.gov/ency/article/003576.htm Acetylcholine receptor antibody To use the sharing features on this page, please enable JavaScript. Acetylcholine receptor antibody is a protein found in the blood of ...

  2. Docking to flexible nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Sander, Tommy; Bruun, Anne T; Balle, Thomas

    2010-01-01

    Computational docking to nicotinic acetylcholine receptors (nAChRs) and other members of the Cys-loop receptor family is complicated by the flexibility of the so-called C-loop. As observed in the large number of published crystal structures of the acetylcholine binding protein (AChBP), a structural...

  3. Neuromodulation: acetylcholine and memory consolidation.

    Science.gov (United States)

    Hasselmo

    1999-09-01

    Clinical and experimental evidence suggests that hippocampal damage causes more severe disruption of episodic memories if those memories were encoded in the recent rather than the more distant past. This decrease in sensitivity to damage over time might reflect the formation of multiple traces within the hippocampus itself, or the formation of additional associative links in entorhinal and association cortices. Physiological evidence also supports a two-stage model of the encoding process in which the initial encoding occurs during active waking and deeper consolidation occurs via the formation of additional memory traces during quiet waking or slow-wave sleep. In this article I will describe the changes in cholinergic tone within the hippocampus in different stages of the sleep-wake cycle and will propose that these changes modulate different stages of memory formation. In particular, I will suggest that the high levels of acetylcholine that are present during active waking might set the appropriate dynamics for encoding new information in the hippocampus, by partially suppressing excitatory feedback connections and so facilitating encoding without interference from previously stored information. By contrast, the lower levels of acetylcholine that are present during quiet waking and slow-wave sleep might release this suppression and thereby allow a stronger spread of activity within the hippocampus itself and from the hippocampus to the entorhinal cortex, thus facilitating the process of consolidation of separate memory traces.

  4. Antagonism of acetylcholine by adrenaline antagonists

    Science.gov (United States)

    Benfey, B. G.; Grillo, S. A.

    1963-01-01

    Phenoxybenzamine antagonized the inhibitory action of acetylcholine on the guinea-pig isolated atrium. The antagonism was slow in onset, very slowly reversible, and could be overcome by increased concentrations of acetylcholine. In contrast, atropine inhibited the action of acetylcholine quickly, and the effect disappeared soon after withdrawal. The pA10 of phenoxybenzamine (2 hr of contact) was 6.8, and that of atropine (30 min of contact) was 8.4. In the presence of atropine phenoxybenzamine did not exert a slowly reversible antagonism, and the dose-ratio of acetylcholine returned to normal soon after withdrawal of both drugs. Phenoxybenzamine also antagonized acetylcholine in the guinea-pig isolated ileum, but with higher concentrations acetylcholine did not overcome the antagonism. The pA10 (60 min of contact) was 6.6. The pA10 of chlorpromazine in the atrium (2 hr of contact) and ileum (60 min of contact) was 5.9. Phentolamine, 2-diethylaminomethylbenzo-1,4-dioxan hydrochloride (883 F), and yohimbine antagonized acetylcholine in the atrium and ileum but required higher concentrations than chlorpromazine. PMID:13967429

  5. The α7 nicotinic acetylcholine receptor complex

    DEFF Research Database (Denmark)

    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 prote......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...

  6. Structural Studies of Nicotinic Acetylcholine Receptors

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Gajhede, Michael; Kastrup, Jette

    2016-01-01

    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...

  7. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Czech Academy of Sciences Publication Activity Database

    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

  8. Plasma concentration of acetylcholine in young women

    International Nuclear Information System (INIS)

    Kawashima, K.; Oohata, H.; Fujimoto, K.; Suzuki, T.

    1987-01-01

    A sensitive and specific radioimmunoassay was applied to the determination of acetylcholine (ACh) in plasma. The concentration of ACh in plasma sampled from 32 young women was 456.1+-53.1 (mean +-S.E.M.) pg/ml. No significant correlations were observed between plasma concentrations of ACh and acetylcholinesterase (AChE) activity, or gonadal hormones. These data demonstrate that an amount of ACh measurable by radioimmunoassay is present in plasma and plasma ACh is not regulated by AChE activity and the menstrual cycle in young women. The origin and physiological as well as pathophysiological significance of ACh in plasma remain to be clarified. 13 refs. (Author)

  9. α-4 subunit of nicotinic acetylcholine receptor polymorphisms exhibit ...

    African Journals Online (AJOL)

    Background: Smoking behavior is influenced by both genetic and environmental factors. Nicotine is the major addictive substance in cigarettes. Nicotinic acetylcholine receptors (nAChRs) are thought to play an important role in nicotine addiction of smokers. One of the genes, α-4 subunit of nicotinic acetylcholine receptor ...

  10. The effect of ketamine on intraspinal acetylcholine release

    DEFF Research Database (Denmark)

    Abelson, Klas S P; Goldkuhl, Renée Röstlinger; Nylund, Anders

    2006-01-01

    The general anaesthetic ketamine affects the central cholinergic system in several manners, but its effect on spinal acetylcholine release, which may be an important transmitter in spinal antinociception, is unknown. This study aimed to investigate the effect of ketamine on spinal acetylcholine...... release. Microdialysis probes were placed intraspinally in male rats, and acetylcholine was quantified with HPLC. Anaesthesia was switched from isoflurane (1.3%) to ketamine (150 mg/kg h), which resulted in a 500% increased acetylcholine release. The increase was attenuated during nicotinic receptor...... blockade (50 microM mecamylamine). The nicotinic receptor agonist epibatidine (175 microM) produced a ten-fold higher relative increase of acetylcholine release during isoflurane anaesthesia compared to ketamine anaesthesia (270% to 27%). Intraspinal administration of ketamine and norketamine both...

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

    Directory of Open Access Journals (Sweden)

    Herman S. Cheung

    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.

  12. The α7 nicotinic acetylcholine receptor complex

    DEFF Research Database (Denmark)

    Thomsen, Morten S; 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 prote......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...... 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-term administration...... with these different types of compounds. Finally, we describe the special case of Aβ1-42 binding to the α7 nAChR, which may pose a unique challenge to drug development of α7 nAChR-specific ligands for Alzheimer's disease. Hopefully, a greater knowledge of the many factors influencing α7 nAChR function as well...

  13. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  14. Alcohol's actions on neuronal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Davis, Tiffany J; de Fiebre, Christopher M

    2006-01-01

    Although it has been known for many years that alcoholism and tobacco addiction often co-occur, relatively little information is available on the biological factors that regulate the co-use and abuse of nicotine and alcohol. In the brain, nicotine acts at several different types of receptors collectively known as nicotinic acetylcholine receptors (nAChRs). Alcohol also acts on at least some of these receptors, enhancing the function of some nAChR subtypes and inhibiting the activity of others. Chronic alcohol and nicotine administration also lead to changes in the numbers of nAChRs. Natural variations (i.e., polymorphisms) in the genes encoding different nAChR subunits may be associated with individual differences in the sensitivity to some of alcohol's and nicotine's effects. Finally, at least one subtype of nAChR may help protect cells against alcohol-induced neurotoxicity.

  15. Impulsive behavior and nicotinic acetylcholine receptors.

    Science.gov (United States)

    Ohmura, Yu; Tsutsui-Kimura, Iku; Yoshioka, Mitsuhiro

    2012-01-01

    Higher impulsivity is thought to be a risk factor for drug addiction, criminal involvement, and suicide. Excessive levels of impulsivity are often observed in several psychiatric disorders including attention-deficit/hyperactivity disorder and schizophrenia. Previous studies have demonstrated that nicotinic acetylcholine receptors (nAChRs) are involved in impulsive behavior. Here, we introduce recent advances in this field and describe the role of the following nAChR-related brain mechanisms in modulating impulsive behavior: dopamine release in the ventral striatum; α4β2 nAChRs in the infralimbic cortex, which is a ventral part of the medial prefrontal cortex (mPFC); and dopamine release in the mPFC. We also suggest several potential therapeutic drugs to address these mechanisms in impulsivity-related disorders and explore future directions to further elucidate the roles of central nAChRs in impulsive behavior.

  16. Inhibition of acetylcholine synthesis in vitro

    International Nuclear Information System (INIS)

    O-Neill, J.J.; Capacio, B.; Doukas, P.H.; Leech, R.; Ricciardi, F.; Sterling, G.H.

    1986-01-01

    In order to better understand diseases that stem from deficiencies in cholinergic activity, reproducible in vitro and in vivo models displaying cholinergic hypofunction are desirable. This necessitates the availability of specific inhibitors. This paper examines the design, synthesis and evaluation of quinuclidinyl compounds with structural features previously reported, but with certain key differences. Structure activity studies with in vitro assay systems are presented. In a few studies, choline was held constant and acetyl-CoA concentration was varied, but with a constant amount of ( 14 C) - acetyl CoA. Acetylcholine synthesis and CO 2 production from labelled glucose were measured in cerebral cortex slices from male rats after decapitation. The nanomoles of ACh and CO 2 produced from ( 14 C) -glucose were calculated from glucose specific activity. Results are presented

  17. Acetylcholine receptors in the human retina

    International Nuclear Information System (INIS)

    Hutchins, J.B.; Hollyfield, J.G.

    1985-01-01

    Evidence for a population of acetylcholine (ACh) receptors in the human retina is presented. The authors have used the irreversible ligand 3 H-propylbenzilylcholine mustard ( 3 H-PrBCM) to label muscarinic receptors. 3 H- or 125 I-alpha-bungarotoxin (alpha-BTx) was used to label putative nicotinic receptors. Muscarinic receptors are apparently present in the inner plexiform layer of the retina. Autoradiographic grain densities are reduced in the presence of saturating concentrations of atropine, quinuclidinyl benzilate or scopolamine; this indicates that 3 H-PrBCM binding is specific for a population of muscarinic receptors in the human retina. Binding sites for radiolabeled alpha-BTx are found predominantly in the inner plexiform layer of the retina. Grain densities are reduced in the presence of d-tubocurarine, indicating that alpha-BTx may bind to a pharmacologically relevant nicotinic ACh receptor. This study provides evidence for cholinergic neurotransmission in the human retina

  18. Methodologic aspects of acetylcholine-evoked relaxation of rabbit aorta

    DEFF Research Database (Denmark)

    Larsen, Kirsten Vendelbo; Nedergaard, Ove A.

    1999-01-01

    The acetylcholine-evoked relaxation of rabbit isolated thoracic aorta precontracted by phenylephrine was studied. Phenylephrine caused a steady contraction that was maintained for 6 h. In the presence of calcium disodium ethylenediaminetetraacetate (EDTA) and ascorbic acid the contraction decreased...

  19. The Ghosts of Acetylcholine : structure-activity relationships of ...

    African Journals Online (AJOL)

    The Ghosts of Acetylcholine : structure-activity relationships of muscle relaxants : registrar communication. ... AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for Librarians · for Authors · FAQ's · More about AJOL · AJOL's ...

  20. Effect of aerosolized acetylcholine on bronchial blood flow.

    Science.gov (United States)

    Charan, N B; Carvalho, P; Johnson, S R; Thompson, W H; Lakshminarayan, S

    1998-08-01

    We studied the effects of aerosolized as well as intravenous infusion of acetylcholine on bronchial blood flow in six anesthetized sheep. Intravenous infusion of acetylcholine, at a dose of 2 microg/kg, increased bronchial blood flow from 45 +/- 15 (SE) to 74 +/- 30 ml/min, and vascular conductance increased by 76 +/- 22%. In contrast, aerosolized acetylcholine at doses of 2 and 20 microg/kg decreased bronchial vascular conductance by approximately 10%. At an aerosolized dose of 200 microg/kg, the bronchial vascular conductance increased by approximately 15%, and there was no further increase in conductance when the aerosolized dose was increased to 2,000 microg/kg. Pretreatment of animals with a nitric oxide synthase inhibitor, Nomega-nitro-L-arginine methyl ester hydrochloride, partially blocked the vasodilatory effects of intravenous acetylcholine and completely blocked the vasodilatory effects of high-dose aerosolized acetylcholine. These data suggest that aerosolized acetylcholine does not readily penetrate the vascular wall of bronchial circulatory system and, therefore, has minimal vasodilatory effects on the bronchial vasculature.

  1. The role of acetylcholine in cocaine addiction.

    Science.gov (United States)

    Williams, Mark J; Adinoff, Bryon

    2008-07-01

    Central nervous system cholinergic neurons arise from several discrete sources, project to multiple brain regions, and exert specific effects on reward, learning, and memory. These processes are critical for the development and persistence of addictive disorders. Although other neurotransmitters, including dopamine, glutamate, and serotonin, have been the primary focus of drug research to date, a growing preclinical literature reveals a critical role of acetylcholine (ACh) in the experience and progression of drug use. This review will present and integrate the findings regarding the role of ACh in drug dependence, with a primary focus on cocaine and the muscarinic ACh system. Mesostriatal ACh appears to mediate reinforcement through its effect on reward, satiation, and aversion, and chronic cocaine administration produces neuroadaptive changes in the striatum. ACh is further involved in the acquisition of conditional associations that underlie cocaine self-administration and context-dependent sensitization, the acquisition of associations in conditioned learning, and drug procurement through its effects on arousal and attention. Long-term cocaine use may induce neuronal alterations in the brain that affect the ACh system and impair executive function, possibly contributing to the disruptions in decision making that characterize this population. These primarily preclinical studies suggest that ACh exerts a myriad of effects on the addictive process and that persistent changes to the ACh system following chronic drug use may exacerbate the risk of relapse during recovery. Ultimately, ACh modulation may be a potential target for pharmacological treatment interventions in cocaine-addicted subjects. However, the complicated neurocircuitry of the cholinergic system, the multiple ACh receptor subtypes, the confluence of excitatory and inhibitory ACh inputs, and the unique properties of the striatal cholinergic interneurons suggest that a precise target of cholinergic

  2. Aspects of dopamine and acetylcholine release induced by glutamate receptors

    International Nuclear Information System (INIS)

    Paes, Paulo Cesar de Arruda

    2002-01-01

    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)

  3. Acetylcholine release and inhibitory interneuron activity in hippocampal CA1

    Directory of Open Access Journals (Sweden)

    A. Rory McQuiston

    2014-09-01

    Full Text Available Acetylcholine release in the central nervous system (CNS has an important role in attention, recall and memory formation. One region influenced by acetylcholine is the hippocampus, which receives inputs from the medial septum and diagonal band of Broca complex (MS/DBB. Release of acetylcholine from the MS/DBB can directly affect several elements of the hippocampus including glutamatergic and GABAergic neurons, presynaptic terminals, postsynaptic receptors and astrocytes. A significant portion of acetylcholine’s effect likely results from the modulation of GABAergic inhibitory interneurons, which have crucial roles in controlling excitatory inputs, synaptic integration, rhythmic coordination of principal neurons and outputs in the hippocampus. Acetylcholine affects interneuron function in large part by altering their membrane potential via muscarinic and nicotinic receptor activation. This minireview describes recent data from mouse hippocampus that investigated changes in CA1 interneuron membrane potentials following acetylcholine release. The interneuron subtypes affected, the receptor subtypes activated, and the potential outcome on hippocampal CA1 network function is discussed.

  4. Cholinergic neurotransmission in human corpus cavernosum. II. Acetylcholine synthesis

    International Nuclear Information System (INIS)

    Blanco, R.; De Tejada, S.; Goldstein, I.; Krane, R.J.; Wotiz, H.H.; Cohen, R.A.

    1988-01-01

    Physiological and histochemical evidence indicates that cholinergic nerves may participate in mediating penile erection. Acetylcholine synthesis and release was studied in isolated human corporal tissue. Human corpus cavernosum incubated with [ 3 H]choline accumulated [ 3 H]choline and synthesized [ 3 H]acethylcholine in an concentration-dependent manner. [ 3 H]Acetylcholine accumulation by the tissue was inhibited by hemicholinium-3, a specific antagonist of the high-affinity choline transport in cholinergic nerves. Transmural electrical field stimulation caused release of [ 3 H]acetylcholine which was significantly diminished by inhibiting neurotransmission with calcium-free physiological salt solution or tetrodotoxin. These observations provide biochemical and physiological evidence for the existence of cholinergic innervation in human corpus cavernosum

  5. Melatonin modulates rat myotube-acetylcholine receptors by inhibiting calmodulin.

    Science.gov (United States)

    de Almeida-Paula, Lidiana Duarte; Costa-Lotufo, Leticia V; Silva Ferreira, Zulma; Monteiro, Amanda Elisa G; Isoldi, Mauro Cesar; Godinho, Rosely O; Markus, Regina P

    2005-11-21

    Melatonin, the pineal gland hormone, modulates alpha-bungarotoxin sensitive nicotinic acetylcholine receptors in sympathetic nerve terminals, cerebellum and chick retina imposing a diurnal variation in functional responses [Markus, R.P., Zago, W.M., Carneiro, R.C., 1996. Melatonin modulation of presynaptic nicotinic acetylcholine receptors in the rat vas deferens. J. Pharmacol. Exp. Ther. 279, 18-22; Markus, R.P., Santos, J.M., Zago, W., Reno, L.A., 2003. Melatonin nocturnal surge modulates nicotinic receptors and nicotine-induced [3HI] glutamate release in rat cerebellum slices. J. Pharmacol. Exp. Ther. 305, 525-530; Sampaio, L.F.S., Hamassaki-Britto, D.E., Markus, R.P., 2005. Influence of melatonin on the development of functional nicotinic acetylcholine receptors in cultured chick retinal cells. Braz. J. Med. Biol. Res. 38, 603-613]. Here we show that in rat myotubes forskolin and melatonin reduced the number of nicotinic acetylcholine receptors expressed in plasma membrane. In addition, these cells expressed melatonin MT1 receptors, which are known to be coupled to G(i)-protein. However, the pharmacological profile of melatonin analogs regarding the reduction in cyclic AMP accumulation and number of nicotinic acetylcholine receptors did not point to a mechanism mediated by activation of G(i)-protein coupled receptors. On the other hand, calmidazolium, a classical inhibitor of calmodulin, reduced in a similar manner both effects. Considering that one isoform of adenylyl cyclase present in rat myotubes is regulated by Ca2+/calmodulin, we propose that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation.

  6. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Directory of Open Access Journals (Sweden)

    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.

  7. Muscarinic acetylcholine receptor expression in aganglionic bowel.

    Science.gov (United States)

    Oue, T; Yoneda, A; Shima, H; Puri, P

    2000-01-01

    In Hirschsprung's disease (HD) there exists an overabundance of acetylcholine (ACh), which in turn stimulates excessive production of the enzyme acetylcholinesterase. Muscarinic ACh receptors (mAChRs) play an important role in smooth-muscle contraction. Recent studies have indicated five different subtypes of mAChRs encoded by five different genes, ml to m5. The purpose of this study was to investigate the expression of each mAChR subtype in aganglionic (AG) colon to further understand the pathophysiology of HD. Entire colon resected at the time of pull-through operation for HD was obtained from 14 patients. Specimens obtained at autopsy from 8 age-matched patients without gastrointestinal disease acted as controls. Frozen sections were used for indirect immunohistochemistry as well as in-situ hybridization. Immunohistochemistry was performed using specific antiserum against each mAChR subtype and in-situ hybridization was performed using specific oligonucleotide probes against ml to m5 subtypes. Messenger RNA (mRNA) was extracted from normoganglionic (NG) and AG bowel of HD patients and normal control bowel. Reverse transcription-polymerase chain reaction was performed to evaluate mRNA levels of each mAChR subtype. To adjust the levels of mRNA expression, a housekeeping gene G3PDH, known to be expressed normally, was used as an internal control. Strong m2 and m3 immunoreactivity was observed in the mucosal layer, smooth-muscle layers, and myenteric plexus of NG bowel, whereas ml immunoreactivity was only detected in the mucosal layer. The most striking finding was the abundance of m3-immunoreactive fibers in muscle layers of NG bowel while there was a total lack of m3 fibers in smooth-muscle of AG bowel. Intense mRNA signals encoding m2 and m3 and to a lesser degree ml were detected in NG bowel, and these signals were weak in AG bowel. Immunoreactivity and mRNA expression of m4 and m5 was not detected in NG or AG bowel. The lack of m3-immunoreactive fibers in the

  8. The effect of acetylcholine on 14C-assimilates translocation of Isatis tinctoria L

    International Nuclear Information System (INIS)

    Yang Chongjun; Tang Feiyu; Zhang Ping; Guo Yuhai

    2004-01-01

    The effects of acetylcholine on 14 C-assimilates translocation are studied with source-channel-sink of Isatis tinctoria L. The experiments show that 0.01 mmol/L treatments of acetylcholine on the phloem, can improve the output of 14 C-assimilates in leaves indicating that acetylcholine enhances the activity of phloem transport. (authors)

  9. Nicotinic Acetylcholine Receptors in the Pathophysiology of Alzheimer's Disease

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Andreasen T., Jesper; Arvaniti, Maria

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) have been pursued for decades as potential molecular targets to treat cognitive dysfunction in Alzheimer's disease (AD) due to their positioning within regions of the brain critical in learning and memory, such as the prefrontal cortex and hippocampus...

  10. Measurement of anti- acetylcholine receptor auto-antibodies in ...

    African Journals Online (AJOL)

    auto-antibodies in myasthenia gravis. K. J. Steenkamp, W. Duim, M. s. Myer,. S. C. K. Malfeld, R. Anderson. Two different acetylcholine receptor (AChR) preparations derived from ... the detection of AChR auto-antibodies in serum specimens from 20 ... 4°C. Thereafter, 1 ml of washing solution (phosphate- buffered saline ...

  11. Cholinergic modulation of dopamine pathways through nicotinic acetylcholine receptors.

    NARCIS (Netherlands)

    de Kloet, S.F.; Mansvelder, H.D.; de Vries, T.J.

    2015-01-01

    Nicotine addiction is highly prevalent in current society and is often comorbid with other diseases. In the central nervous system, nicotine acts as an agonist for nicotinic acetylcholine receptors (nAChRs) and its effects depend on location and receptor composition. Although nicotinic receptors are

  12. EVP-6124, a novel and selective α7 nicotinic acetylcholine receptor partial agonist, improves memory performance by potentiating the acetylcholine response of α7 nicotinic acetylcholine receptors.

    Science.gov (United States)

    Prickaerts, Jos; van Goethem, Nick P; Chesworth, Richard; Shapiro, Gideon; Boess, Frank G; Methfessel, Christoph; Reneerkens, Olga A H; Flood, Dorothy G; Hilt, Dana; Gawryl, Maria; Bertrand, Sonia; Bertrand, Daniel; König, Gerhard

    2012-02-01

    EVP-6124, (R)-7-chloro-N-quinuclidin-3-yl)benzo[b]thiophene-2-carboxamide, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs) that was evaluated here in vitro and in vivo. In binding and functional experiments, EVP-6124 showed selectivity for α7 nAChRs and did not activate or inhibit heteromeric α4β2 nAChRs. EVP-6124 had good brain penetration and an adequate exposure time. EVP-6124 (0.3 mg/kg, p.o.) significantly restored memory function in scopolamine-treated rats (0.1 mg/kg, i.p.) in an object recognition task (ORT). Although donepezil at 0.1 mg/kg, p.o. or EVP-6124 at 0.03 mg/kg, p.o. did not improve memory in this task, co-administration of these sub-efficacious doses fully restored memory. In a natural forgetting test, an ORT with a 24 h retention time, EVP-6124 improved memory at 0.3 mg/kg, p.o. This improvement was blocked by the selective α7 nAChR antagonist methyllycaconitine (0.3 mg/kg, i.p. or 10 μg, i.c.v.). In co-application experiments of EVP-6124 with acetylcholine, sustained exposure to EVP-6124 in functional investigations in oocytes caused desensitization at concentrations greater than 3 nM, while lower concentrations (0.3-1 nM) caused an increase in the acetylcholine-evoked response. These actions were interpreted as representing a co-agonist activity of EVP-6124 with acetylcholine on α7 nAChRs. The concentrations of EVP-6124 that resulted in physiological potentiation were consistent with the free drug concentrations in brain that improved memory performance in the ORT. These data suggest that the selective partial agonist EVP-6124 improves memory performance by potentiating the acetylcholine response of α7 nAChRs and support new therapeutic strategies for the treatment of cognitive impairment. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    DEFF Research Database (Denmark)

    Risveden, Klas; Dick, Kimberly A; Bhand, Sunil

    2010-01-01

    A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN(x)-covered w......A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on Si......N(x)-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area...

  14. Long release latencies are increased by acetylcholine at frog endplate

    Czech Academy of Sciences Publication Activity Database

    Samigullin, D.; Bukharaeva, E. A.; Nikolsky, E.; Adámek, S.; Vyskočil, František

    2003-01-01

    Roč. 52, č. 4 (2003), s. 475-480 ISSN 0862-8408 R&D Projects: GA ČR GA305/02/1333; GA ČR GA202/02/1213 Grant - others:RFBR(RU) 02/04/48901 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003 Keywords : quantal release * acetylcholine * synaptic latency Subject RIV: ED - Physiology Impact factor: 0.939, year: 2003

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

    Directory of Open Access Journals (Sweden)

    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

  16. Branched nanotrees with immobilized acetylcholine esterase for nanobiosensor applications

    Energy Technology Data Exchange (ETDEWEB)

    Risveden, Klas; Bhand, Sunil; Danielsson, Bengt [Department of Pure and Applied Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund (Sweden); Dick, Kimberly A; Samuelson, Lars [Solid State Physics, Lund University, Box 118, S-22100 Lund (Sweden); Rydberg, Patrik, E-mail: Kimberly.Dick@ftf.lth.se [Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

    2010-02-05

    A novel lab-on-a-chip nanotree enzyme reactor is demonstrated for the detection of acetylcholine. The reactors are intended for use in the RISFET (regional ion sensitive field effect transistor) nanosensor, and are constructed from gold-tipped branched nanorod structures grown on SiN{sub x}-covered wafers. Two different reactors are shown: one with simple, one-dimensional nanorods and one with branched nanorod structures (nanotrees). Significantly higher enzymatic activity is found for the nanotree reactors than for the nanorod reactors, most likely due to the increased gold surface area and thereby higher enzyme binding capacity. A theoretical calculation is included to show how the enzyme kinetics and hence the sensitivity can be influenced and increased by the control of electrical fields in relation to the active sites of enzymes in an electronic biosensor. The possible effects of electrical fields employed in the RISFET on the function of acetylcholine esterase is investigated using quantum chemical methods, which show that the small electric field strengths used are unlikely to affect enzyme kinetics. Acetylcholine esterase activity is determined using choline oxidase and peroxidase by measuring the amount of choline formed using the chemiluminescent luminol reaction.

  17. Interactions of calcium homeostasis, acetylcholine metabolism, behavior and 3, 4-diaminopyridine during aging

    International Nuclear Information System (INIS)

    Gibson, G.E.; Peterson, C.

    1986-01-01

    Acetylcholine synthesis declines with aging in both whole brain and in various brain regions. Since neither enzyme activities nor acetylcholine concentrations, accurately reflect the dynamics of the cholinergic system, in vivo acetylcholine formation was measured. Incorporation of U-C 14-glucose of 2 H 4 choline into whole brain acetylcholine decreases from 100% (3 months) in two strains of mice. The diminished synthesis is apparently not due to a lack of precursor availability because U- C 14-glucose and 2 H 4 choline entry into the brain is similar at all ages. It is shown that altered brain calcium homeostasis during aging may underlie the deficits in acetylcholine metabolism, as well as those in behavior. Diminished calcium uptake during aging parallels the decline in the calcium dependent release of acetylcholine

  18. Why we forget our dreams: Acetylcholine and norepinephrine in wakefulness and REM sleep.

    Science.gov (United States)

    Becchetti, Andrea; Amadeo, Alida

    2016-01-01

    The ascending fibers releasing norepinephrine and acetylcholine are highly active during wakefulness. In contrast, during rapid-eye-movement sleep, the neocortical tone is sustained mainly by acetylcholine. By comparing the different physiological features of the norepinephrine and acetylcholine systems in the light of the GANE (glutamate amplifies noradrenergic effects) model, we suggest how to interpret some functional differences between waking and rapid-eye-movement sleep.

  19. Contrasting actions of philanthotoxin-343 and philanthotoxin-(12) on human muscle nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Brier, Tim J; Mellor, Ian R; Tikhonov, Denis B

    2003-01-01

    Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle nicotinic acetylcholine receptors (nAChR) by the philanthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation-dependent, nonc......Whole-cell recordings and outside-out patch recordings from TE671 cells were made to investigate antagonism of human muscle nicotinic acetylcholine receptors (nAChR) by the philanthotoxins, PhTX-343 and PhTX-(12). When coapplied with acetylcholine (ACh), PhTX-343 caused activation...

  20. Back to the future: Rational maps for exploring acetylcholine receptor space and time.

    Science.gov (United States)

    Tessier, Christian J G; Emlaw, Johnathon R; Cao, Zhuo Qian; Pérez-Areales, F Javier; Salameh, Jean-Paul J; Prinston, Jethro E; McNulty, Melissa S; daCosta, Corrie J B

    2017-11-01

    Global functions of nicotinic acetylcholine receptors, such as subunit cooperativity and compatibility, likely emerge from a network of amino acid residues distributed across the entire pentameric complex. Identification of such networks has stymied traditional approaches to acetylcholine receptor structure and function, likely due to the cryptic interdependency of their underlying amino acid residues. An emerging evolutionary biochemistry approach, which traces the evolutionary history of acetylcholine receptor subunits, allows for rational mapping of acetylcholine receptor sequence space, and offers new hope for uncovering the amino acid origins of these enigmatic properties. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Acetylcholine synthesis and possible functions during sea urchin development

    Directory of Open Access Journals (Sweden)

    C Angelini

    2009-06-01

    Full Text Available Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh, the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 ?M eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the

  2. Impact of acetylcholine and nicotine on human osteoclastogenesis in vitro.

    Science.gov (United States)

    Ternes, Sebastian; Trinkaus, Katja; Bergen, Ivonne; Knaack, Sven; Gelinsky, Michael; Kilian, Olaf; Heiss, Christian; Lips, Katrin Susanne

    2015-11-01

    Recent studies showed that the non-neuronal cholinergic system (NNCS) is taking part in bone metabolism. Most studies investigated its role in osteoblasts, but up to now, the involvement of the NNCS in human osteoclastogenesis remains relatively unclear. Thus, aim of the present study was to determine whether the application of acetylcholine (ACh, 10(−4) M), nicotine (10(−6) M), mineralized collagen membranes or brain derived neurotrophic factor (BDNF, 40 ng/mL) influences the mRNA regulation of molecular components of the NNCS and the neurotrophin family during osteoclastogenesis. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of young healthy donors (n = 8) and incubated with bone fragments and osteoclast differentiation media for 21 days. All the results are based on the measurement of RNA. Real-time RT-PCR analysis demonstrated a down-regulation of nicotinic acetylcholine receptor (nAChR) subunit α2 and muscarinic acetylcholine receptor (mAChR) M3by osteoclastogenesis while BDNF mRNA expression was not regulated. Application of ACh, nicotine, BDNF or collagen membranes did not affect osteoclastic differentiation.No regulation was detected for nAChR subunit α7, tropomyosin-related kinase receptor B (TrkB), and cholineacetyl transferase (ChAT). Taken together, we assume that the transcriptional level of osteoclastogenesis of healthy young humans is not regulated by BDNF, ACh, and nicotine. Thus, these drugs do not seem to worsen bone degradation and might therefore be suitable as modulators of bone substitution materials if having a positive effect on bone formation.

  3. Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Ahring, Philip K; Christensen, Jeppe K

    2011-01-01

    The neuronal a4ß2 nicotinic acetylcholine receptors exist as two distinct subtypes, (a4)(2)(ß2)(3) and (a4)(3)(ß2)(2), and biphasic responses to acetylcholine and other agonists have been ascribed previously to coexistence of these two receptor subtypes. We offer a novel and radical explanation...

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

    DEFF Research Database (Denmark)

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

    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...

  5. Characterization of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance

    NARCIS (Netherlands)

    Hoekstra, R.; Visser, A.; Wiley, L. J.; Weiss, A. S.; Sangster, N. C.; Roos, M. H.

    1997-01-01

    The anthelminitic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the

  6. Characterisation of an acetylcholine receptor gene of Haemonchus contortus in relation to levamisole resistance

    NARCIS (Netherlands)

    Hoekstra, R.; Visser, A.; Wiley, L.; Weiss, A.S.; Sangster, N.C.; Roos, M.H.

    1997-01-01

    The anthelmintic drug levamisole is thought to bind to nicotinic acetylcholine receptors of nematodes. It is possible that resistance to this drug is associated with either a change in binding characteristics or a reduction in the number of nicotinic acetylcholine receptors. Therefore, the molecular

  7. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    International Nuclear Information System (INIS)

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced 155 Eu: 3+ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor

  8. Organophosphate acetylcholine esterase inhibitor poisoning from a home-made shampoo.

    Science.gov (United States)

    Sadaka, Yair; Broides, Arnon; Tzion, Raffi Lev; Lifshitz, Matitiahu

    2011-07-01

    Organophosphate acetylcholine esterase inhibitor poisoning is a major health problem in children. We report an unusual cause of organophosphate acetylcholine esterase inhibitor poisoning. Two children were admitted to the pediatric intensive care unit due to organophosphate acetylcholine esterase inhibitor poisoning after exposure from a home-made shampoo that was used for the treatment of head lice. Owing to no obvious source of poisoning, the diagnosis of organophosphate acetylcholine esterase inhibitor poisoning in one of these patients was delayed. Both patients had an uneventful recovery. Organophosphate acetylcholine esterase inhibitor poisoning from home-made shampoo is possible. In cases where the mode of poisoning is unclear, direct questioning about the use of home-made shampoo is warranted, in these cases the skin and particularly the scalp should be rinsed thoroughly as soon as possible.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  10. Nucleus Accumbens Acetylcholine Receptors Modulate Dopamine and Motivation.

    Science.gov (United States)

    Collins, Anne L; Aitken, Tara J; Greenfield, Venuz Y; Ostlund, Sean B; Wassum, Kate M

    2016-11-01

    Environmental reward-predictive cues can motivate reward-seeking behaviors. Although this influence is normally adaptive, it can become maladaptive in disordered states, such as addiction. Dopamine release in the nucleus accumbens core (NAc) is known to mediate the motivational impact of reward-predictive cues, but little is known about how other neuromodulatory systems contribute to cue-motivated behavior. Here, we examined the role of the NAc cholinergic receptor system in cue-motivated behavior using a Pavlovian-to-instrumental transfer task designed to assess the motivating influence of a reward-predictive cue over an independently-trained instrumental action. Disruption of NAc muscarinic acetylcholine receptor activity attenuated, whereas blockade of nicotinic receptors augmented cue-induced invigoration of reward seeking. We next examined a potential dopaminergic mechanism for this behavioral effect by combining fast-scan cyclic voltammetry with local pharmacological acetylcholine receptor manipulation. The data show evidence of opposing modulation of cue-evoked dopamine release, with muscarinic and nicotinic receptor antagonists causing suppression and augmentation, respectively, consistent with the behavioral effects of these manipulations. In addition to demonstrating cholinergic modulation of naturally-evoked and behaviorally-relevant dopamine signaling, these data suggest that NAc cholinergic receptors may gate the expression of cue-motivated behavior through modulation of phasic dopamine release.

  11. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    Science.gov (United States)

    Cohen, Emiliano; Chatzigeorgiou, Marios; Husson, Steven J.; Steuer-Costa, Wagner; Gottschalk, Alexander; Schafer, William R.; Treinin, Millet

    2014-01-01

    Polymodal nociceptors sense and integrate information on injurious mechanical, thermal, and chemical stimuli. Chemical signals either activate nociceptors or modulate their responses to other stimuli. One chemical known to activate or modulate responses of nociceptors is acetylcholine (ACh). Across evolution nociceptors express subunits of the nicotinic acetylcholine receptor (nAChR) family, a family of ACh-gated ion channels. The roles of ACh and nAChRs in nociceptor function are, however, poorly understood. Caenorhabditis elegans polymodal nociceptors, PVD, express nAChR subunits on their sensory arbor. Here we show that mutations reducing ACh synthesis and mutations in nAChR subunits lead to defects in PVD function and morphology. A likely cause for these defects is a reduction in cytosolic calcium measured in ACh and nAChR mutants. Indeed, overexpression of a calcium pump in PVD mimics defects in PVD function and morphology found in nAChR mutants. Our results demonstrate, for the first time, a central role for nAChRs and ACh in nociceptor function and suggest that calcium permeating via nAChRs facilitates activity of several signaling pathways within this neuron. PMID:24518198

  12. Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

    Science.gov (United States)

    Grewaal, D. S.; Quastel, J. H.

    1973-01-01

    1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke–bicarbonate–[U-14C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K+-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na+ and Ca2+ in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K+, ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na+). 2. Stimulation by K+ of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na+, but some synthesis and release of radioactive acetylcholine occurred in the absence of Na+ as well as in the absence of Ca2+. 3. The Na+ dependence of K+-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na+ lack, and partly due to the suppression of the activity of the Na+ pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca2+ from the incubation medium. It completely blocked K+ stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K+, sodium l-glutamate and lack of Ca2+ on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took

  13. Acetylcholine content and viability of cholinergic neurons are influenced by the activity of protein histidine phosphatase

    Science.gov (United States)

    2012-01-01

    Background The first mammalian protein histidine phosphatase (PHP) was discovered in the late 90s of the last century. One of the known substrates of PHP is ATP-citrate lyase (ACL), which is responsible - amongst other functions - for providing acetyl-CoA for acetylcholine synthesis in neuronal tissues. It has been shown in previous studies that PHP downregulates the activity of ACL by dephosphorylation. According to this our present work focused on the influence of PHP activity on the acetylcholine level in cholinergic neurons. Results The amount of PHP in SN56 cholinergic neuroblastoma cells was increased after overexpression of PHP by using pIRES2-AcGFP1-PHP as a vector. We demonstrated that PHP overexpression reduced the acetylcholine level and induced cell death. The acetylcholine content of SN56 cells was measured by fast liquid chromatography-tandem mass spectrometry method. Overexpression of the inactive H53A-PHP mutant also induced cell damage, but in a significantly reduced manner. However, this overexpression of the inactive PHP mutant did not change the acetylcholine content of SN56 cells significantly. In contrast, PHP downregulation, performed by RNAi-technique, did not induce cell death, but significantly increased the acetylcholine content in SN56 cells. Conclusions We could show for the first time that PHP downregulation increased the acetylcholine level in SN56 cells. This might be a potential therapeutic strategy for diseases involving cholinergic deficits like Alzheimer's disease. PMID:22436051

  14. Dietary restriction of choline reduces hippocampal acetylcholine release in rats: in vivo microdialysis study.

    Science.gov (United States)

    Nakamura, A; Suzuki, Y; Umegaki, H; Ikari, H; Tajima, T; Endo, H; Iguchi, A

    2001-12-01

    We fed rats with a diet deficient in choline for 12 weeks and studied how dietary choline deficiency affected their behavior and their ability to release acetylcholine in discrete regions of rat brain using step-through passive avoidance task and in vivo microdialysis. In comparison with the control, rats fed the choline-deficient diet showed poorer retention of nociceptive memory in the passive avoidance task. Average choline level in cerebrospinal fluid in the choline-deficient group was significantly less (33.1%) than that of control rats. In vivo microdialysis showed no difference in the pattern of acetylcholine release enhanced by intraperitoneal administration of scopolamine hydrochloride (2 mg/kg) in the striatum between the two groups, whereas in the hippocampus, the maximum and subsequent increase of acetylcholine from the baseline by scopolamine injection was significantly lower in the choline-deficient group than in the control. From the results of our study, we speculate that long-term dietary restriction of choline can affect extra- and intracellular sources of substrates required for acetylcholine synthesis, and eventually limit the ability to release acetylcholine in the hippocampus. Reduced capacity to release acetylcholine in the hippocampus implies that the mechanism, maintaining acetylcholine synthesis on increased neuronal demand, may vary in discrete regions of the brain in response to dietary manipulation. The vulnerability of the mechanism in the hippocampus to dietary choline restriction is indicated by impaired mnemonic performance we observed.

  15. Modulation of the effect of acetylcholine on insulin release by the membrane potential of B cells

    International Nuclear Information System (INIS)

    Hermans, M.P.; Schmeer, W.; Henquin, J.C.

    1987-01-01

    Mouse islets were used to test the hypothesis that the B cell membrane must be depolarized for acetylcholine to increase insulin release. The resting membrane potential of B cells (at 3 mM glucose) was slightly decreased (5 mV) by acetylcholine, but no electrical activity appeared. This depolarization was accompanied by a Ca-independent acceleration of 86 Rb and 45 Ca efflux but no insulin release. When the B cell membrane was depolarized by a stimulatory concentration of glucose (10 mM), acetylcholine potentiated electrical activity, accelerated 86 Rb and 45 Ca efflux, and increased insulin release. This latter effect, but not the acceleration of 45 Ca efflux, was totally dependent on extracellular Ca. If glucose-induced depolarization of the B cell membrane was prevented by diazoxide, acetylcholine lost all effects but those produced at low glucose. In contrast, when the B cell membrane was depolarized by leucine or tolbutamide (at 3 mM glucose), acetylcholine triggered a further depolarization with appearance of electrical activity, accelerated 86 Rb and 45 Ca efflux, and stimulated insulin release. Acetylcholine produced similar effects (except for electrical activity) in the presence of high K or arginine which, unlike the above test agents, depolarize the B cell membrane by a mechanism other than a decrease in K+ permeability. Omission of extracellular Ca abolished the releasing effect of acetylcholine under all conditions but only partially decreased the stimulation of 45 Ca efflux. The results show thus that acetylcholine stimulation of insulin release does not result from mobilization of cellular Ca but requires that the B cell membrane be sufficiently depolarized to reach the threshold potential where Ca channels are activated. This may explain why acetylcholine alone does not initiate release but becomes active in the presence of a variety of agents

  16. Functional interaction between Lypd6 and nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Arvaniti, Maria; Jensen, Majbrit M; Soni, Neeraj

    2016-01-01

    Nicotinic acetylcholine receptors (nAChRs) affect multiple physiological functions in the brain and their functions are modulated by regulatory proteins of the Lynx family. Here, we report for the first time a direct interaction of the Lynx protein LY6/PLAUR domain-containing 6 (Lypd6) with n...... brain. Additionally, soluble recombinant Lypd6 protein attenuates nicotine-induced hippocampal inward currents in rat brain slices and decreases nicotine-induced extracellular signal-regulated kinase phosphorylation in PC12 cells, suggesting that binding of Lypd6 is sufficient to inhibit n......AChR-mediated intracellular signaling. We further show that perinatal nicotine exposure in rats (4 mg/kg/day through minipumps to dams from embryonic day 7 to post-natal day 21) significantly increases Lypd6 protein levels in the hippocampus in adulthood, which did not occur after exposure to nicotine in adulthood only. Our...

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

    International Nuclear Information System (INIS)

    Zeitlhofer, J.; Maida, E.M.; Mamoli, B.; Mayr, N.

    1986-01-01

    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)

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

    NARCIS (Netherlands)

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

    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

  19. Electrolyte and protein secretion by the perfused rabbit mandibular gland stimulated with acetylcholine or catecholamines

    DEFF Research Database (Denmark)

    Case, R M; Conigrave, A D; Novak, I

    1980-01-01

    stimulation, the rate of protein secretion fell off much faster than the rate of fluid secretion.7. The beta-adrenergic agonist isoproterenol evoked a fluid secretory response only equal to about 5% of that evoked by acetylcholine, but still the response declined during continued stimulation. The electrolyte...... composition of isoproterenol-evoked saliva was vastly different from that evoked by acetylcholine, being particularly rich in K and HCO(3). The isoproterenol-evoked saliva was also extremely rich in protein so that the total protein secretion evoked by isoproterenol was much greater than that evoked...... unstimulated or evoked by acetylcholine or eserine, could be blocked completely by atropine.4. During prolonged stimulation with acetylcholine, the fluid secretory response declined rapidly over a period of about 15 min from an initial high value to a much lower plateau value. After 3 or more hours...

  20. Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

    Energy Technology Data Exchange (ETDEWEB)

    Zharkovskii, A.M.; Langel, Yu.L.; Chereshka, K.S.; Zharkovskaya, T.A.

    1987-08-01

    The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

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

    OpenAIRE

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-01-01

    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 musc...

  2. Mechanics of channel gating of the nicotinic acetylcholine receptor.

    Directory of Open Access Journals (Sweden)

    Xinli Liu

    2008-01-01

    Full Text Available The nicotinic acetylcholine receptor (nAChR is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. To address the mechanical fundamentals of nAChR gating, both conventional molecular dynamics (CMD and steered rotation molecular dynamics (SRMD simulations have been conducted on the cryo-electron microscopy (cryo-EM structure of nAChR embedded in a dipalmitoylphosphatidylcholine (DPPC bilayer and water molecules. A 30-ns CMD simulation revealed a collective motion amongst C-loops, M1, and M2 helices. The inward movement of C-loops accompanying the shrinking of acetylcholine (ACh binding pockets induced an inward and upward motion of the outer beta-sheet composed of beta9 and beta10 strands, which in turn causes M1 and M2 to undergo anticlockwise motions around the pore axis. Rotational motion of the entire receptor around the pore axis and twisting motions among extracellular (EC, transmembrane (TM, and intracellular MA domains were also detected by the CMD simulation. Moreover, M2 helices undergo a local twisting motion synthesized by their bending vibration and rotation. The hinge of either twisting motion or bending vibration is located at the middle of M2, possibly the gate of the receptor. A complementary twisting-to-open motion throughout the receptor was detected by a normal mode analysis (NMA. To mimic the pulsive action of ACh binding, nonequilibrium MD simulations were performed by using the SRMD method developed in one of our laboratories. The result confirmed all the motions derived from the CMD simulation and NMA. In addition, the SRMD simulation indicated that the channel may undergo an open-close (O C motion. The present MD simulations explore the structural dynamics of the receptor under its

  3. Partial neuromuscular blockade in humans enhances muscle blood flow during exercise independently of muscle oxygen uptake and acetylcholine receptor blockade

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Krustrup, Peter; Iaia, F Marcello

    2009-01-01

    This study examined the role of acetylcholine for skeletal muscle blood flow during exercise by use of the competitive neuromuscular blocking agent cisatracurium in combination with the acetylcholine receptor blocker glycopyrrone. Nine healthy male subjects performed a 10-min bout of one-legged k......This study examined the role of acetylcholine for skeletal muscle blood flow during exercise by use of the competitive neuromuscular blocking agent cisatracurium in combination with the acetylcholine receptor blocker glycopyrrone. Nine healthy male subjects performed a 10-min bout of one...... conductance during exercise, events that are not associated with either acetylcholine or an increased oxygen demand. The results do not support an essential role for acetylcholine, released form the neuromuscular junction, in exercise hyperaemia or for the enhanced blood flow during neuromuscular blockade....... The enhanced exercise hyperemia during partial neuromuscular blockade may be related to a greater recruitment of fast-twitch muscle fibres. Key words: blood flow, neuromuscular blockade, exercise, skeletal muscle....

  4. Dopamine D(1) receptor-mediated control of striatal acetylcholine release by endogenous dopamine.

    Science.gov (United States)

    Acquas, E; Di Chiara, G

    1999-10-27

    The role of dopamine D(1) and D(2) receptors in the control of acetylcholine release in the dorsal striatum by endogenous dopamine was investigated by monitoring with microdialysis the effect of the separate or combined administration of the dopamine D(1) receptor antagonist, SCH 39166 ¿(-)-trans-6,7,7a,8,9, 13b-exahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]-nap hto-[2, 1b]-azepine hydrochloride¿ (50 microg/kg subcutaneous (s.c.)), of the dopamine D(2)/D(3) receptor agonist, quinpirole (trans-(-)-4aR, 4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo-(3,4-g)-quinoline hydrochloride) (5 and 10 microg/kg s.c.), and of the D(3) receptor selective agonist, PD 128,907 [S(+)-(4aR,10bR)-3,4,4a, 10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano-[4,3-b]-1,4-oxazin -9-ol hydrochloride] (50 microg/kg s.c.), on in vivo dopamine and acetylcholine release. Microdialysis was performed with a Ringer containing low concentrations (0.01 microM) of the acetylcholinesterase inhibitor, neostigmine. Quinpirole (10 microg/kg s.c.) decreased striatal dopamine and acetylcholine release. Administration of PD 128,907 (50 microg/kg) decreased dopamine but failed to affect acetylcholine release. SCH 39166 (50 microg/kg s.c.) stimulated dopamine release and reduced acetylcholine release. Pretreatment with quinpirole reduced (5 microg/kg s.c.) or completely prevented (10 microg/kg s.c.) the stimulation of dopamine release elicited by SCH 39166 (50 microg/kg s.c.); on the other hand, pretreatment with quinpirole (5 and 10 microg/kg) potentiated the reduction of striatal acetylcholine release induced by SCH 39166 (50 microg/kg s.c.). Similarly, pretreatment with PD 128,907 (50 microg/kg) which prevented the increase of dopamine release induced by SCH 39166 (50 microg/kg), potentiated the reduction of striatal acetylcholine transmission elicited by SCH 39166. Thus, pretreatment with low doses of quinpirole or PD 128,907 influences in opposite manner the effect of SCH 39166 on striatal dopamine and

  5. Menthol binding and inhibition of α7-nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Abrar Ashoor

    Full Text Available Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca(2+-dependent Cl(- channels, since menthol inhibition remained unchanged by intracellular injection of the Ca(2+ chelator BAPTA and perfusion with Ca(2+-free bathing solution containing Ba(2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [(125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca(2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner.

  6. Electrically induced release of acetylcholine from denervated Schwann cells.

    Science.gov (United States)

    Dennis, M J; Miledi, R

    1974-03-01

    1. Focal electrical stimulation of Schwann cells at the end-plates of denervated frog muscles elicited slow depolarizations of up to 30 mV in the muscle fibres. This response is referred to as a Schwann-cell end-plate potential (Schwann-e.p.p.).2. Repeated stimulation sometimes evoked further Schwann-e.p.p.s, but they were never sustained for more than 30 pulses. Successive e.p.p.s varied in amplitude and time course independently of the stimulus.3. The Schwann-e.p.p.s were reversibly blocked by curare, suggesting that they result from a release of acetylcholine (ACh) by the Schwann cells.4. ACh release by electrical stimulation did not seem to occur in quantal form and was not dependent on the presence of calcium ions in the external medium; nor was it blocked by tetrodotoxin.5. Stimulation which caused release of ACh also resulted in extensive morphological disruption of the Schwann cells, as seen with both light and electron microscopy.6. It is concluded that electrical stimulation of denervated Schwann cells causes break-down of the cell membrane and releases ACh, presumably in molecular form.

  7. Cost-benefit decision circuitry: proposed modulatory role for acetylcholine.

    Science.gov (United States)

    Fobbs, Wambura C; Mizumori, Sheri J Y

    2014-01-01

    In order to select which action should be taken, an animal must weigh the costs and benefits of possible outcomes associate with each action. Such decisions, called cost-benefit decisions, likely involve several cognitive processes (including memory) and a vast neural circuitry. Rodent models have allowed research to begin to probe the neural basis of three forms of cost-benefit decision making: effort-, delay-, and risk-based decision making. In this review, we detail the current understanding of the functional circuits that subserve each form of decision making. We highlight the extensive literature by detailing the ability of dopamine to influence decisions by modulating structures within these circuits. Since acetylcholine projects to all of the same important structures, we propose several ways in which the cholinergic system may play a local modulatory role that will allow it to shape these behaviors. A greater understanding of the contribution of the cholinergic system to cost-benefit decisions will permit us to better link the decision and memory processes, and this will help us to better understand and/or treat individuals with deficits in a number of higher cognitive functions including decision making, learning, memory, and language. © 2014 Elsevier Inc. All rights reserved.

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

    International Nuclear Information System (INIS)

    Large, T.H.; Cho, N.J.; De Mello, F.G.; Klein, W.L.

    1985-01-01

    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

  9. Actions of piperidine alkaloid teratogens at fetal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Green, Benedict T; Lee, Stephen T; Panter, Kip E; Welch, Kevin D; Cook, Daniel; Pfister, James A; Kem, William R

    2010-01-01

    Teratogenic alkaloids are found in many species of plants including Conium maculatum L., Nicotiana glauca, Nicotiana tabaccum, and multiple Lupinus spp. Fetal musculoskeletal defects produced by alkaloids from these plants include arthrogyropisis, scoliosis, torticollis, kyposis, lordosis, and cleft palate. A pharmacodynamic comparison of the alkaloids ammodendrine, anabasine, anabaseine, anagyrine, and coniine in SH-SY5Y cells and TE-671 cells was made. These alkaloids and their enantiomers were more effective in depolarizing TE-671 cells which express the human fetal-muscle type nicotinic acetylcholine receptor (nAChR) relative to SH-SY5Y cells which predominately express autonomic nAChRs. The rank order of potency in TE-671 cells was: anabaseine>(+)-anabasine>(-)-anabasine > (+/-)-anabasine>anagyrine>(-)-coniine > (+/-)-coniine>(+)-coniine>(+/-)-ammodendrine>(+)-ammodendrine. The rank order potency in SH-SY5Y cells was: anabaseine>(+)-anabasine>(-)-coniine>(+)-coniine>(+)-ammodendrine>anagyrine>(-)-anabasine>(+/-)-coniine>(+/-)-anabasine>(-)-ammodendrine. The actions of these alkaloids at nAChRs in both cell lines could be distinguished by their maximum effects in depolarizing cell membrane potential. The teratogenic action of these compounds may be related to their ability to activate and subsequently desensitize nAChRs.

  10. Effects of two oxadiazolidinones on cholinesterases and acetylcholine receptors

    International Nuclear Information System (INIS)

    Bakry, N.; Lockyer, S.; Sherby, S.; Eldefrawi, A.; Eldefrawi, M.

    1986-01-01

    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( 3 H)-one (DBOX) and 3-(2-methoxyphenyl)-5-methoxy-1,3,4-oxadiazol-2( 3 H)-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 [ 125 I] α bungarotoxin and [ 3 H]perhydrohistrionicotoxin to Torpedo nicotinic acetylcholine receptors, but did not affect binding of [ 3 H]quinuclidinyl benzilate to rat brain muscarinic receptors

  11. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    International Nuclear Information System (INIS)

    Herholz, Karl

    2008-01-01

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  12. Muscarinic acetylcholine receptors: location of the ligand binding site

    International Nuclear Information System (INIS)

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-01-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

  13. Transmembrane topology of the acetylcholine receptor examined in reconstituted vesicles

    International Nuclear Information System (INIS)

    McCrea, P.D.

    1987-01-01

    Each of the five acetylcholine receptor (AChR) subunits, α 2 β-γδ, is believed to have the same number of transmembrane crossing and to share the same general folding pattern. AChR isolated from the electric organ of electric fish is predominantly dimeric. We have used this bridge as a marker for the C-terminus of the δ subunit, and presumably that of the other subunits in addition. The disulfide's accessibility to hydrophilic reductants, principally glutathione (GSH), was tested in a reconstituted vesicle system. The reduction of the δ-δ desulfide, as evidenced by the transition of AChrR dimers to monomers, was quantitatively monitored on velocity sedimentation sucrose gradients. Alternatively, the reduction of δ 2 to δ was followed by employing non-reducing SDS-PAGE. Reductants such as GSH were able to access the bridge in intact right-side-out vesicles. No acceleration of this process was evident when the vesicles were disrupted by freeze-thaw or by detergents. Control experiments which determined the rate of reduction of entrapped diphtheria toxin, or that of 3 H-GSH efflux, demonstrated that intact reconstituted vesicles provide an adequate permeability barrier to GSH access of their intravesicular space

  14. Biochemical and immunological studies of the Muscarinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Gainer, M.W.

    1985-01-01

    Muscarinic acetylcholine receptors were solubilized from bovine brain membranes with 3[3-cholamidopropyl)dimethylammonio]propanesulfonate (CHAPS). A combination of 10 mM CHAPS and 1 M NaCl solubilized 15-40% of the specific receptor binding sites from these membranes. The solubilized receptors displayed high affinity binding of the muscarinic antagonist, [ 3 H]quinuclidinyl benzilate with a K/sub D/ = 300 pM. In addition, the solubilized and retained guanyl nucleotide regulation of agonist binding characteristic of membrane bound receptors. Gel filtration experiments showed that solubilized receptors from cortex and cerebellum had different elution profiles. Analysis by sucrose density gradient centrifugation showed that receptors in the lower molecular weight peak sedimented with a coefficient of 5S. Receptors in the larger molecular weight peak sedimented to the bottom of the gradient. Attempts to purify receptors by chromatography on propylbenzilycholine Sepharose were unsuccessful. The technique used to attach the ligand to the solid support, however, was used to synthesize a PrBCM-BSA conjugate and the conjugate used as an antigen in the production of anti-ligand antibodies. Two anti-PrBCM monoclonal antibodies were isolated that recognize muscarinic but not nicotinic cholinergic ligands. The abilities of the antibodies to recognize other muscarinic ligands indicated the antibodies recognized a portion of PrBCM involved in binding to the receptor. Construction of an antibody affinity resin resulted in the purification of this fragment a minimum of 170 fold

  15. Early Life Stress, Nicotinic Acetylcholine Receptors and Alcohol Use Disorders

    Directory of Open Access Journals (Sweden)

    Joan Y. Holgate

    2015-06-01

    Full Text Available Stress is a major driving force in alcohol use disorders (AUDs. It influences how much one consumes, craving intensity and whether an abstinent individual will return to harmful alcohol consumption. We are most vulnerable to the effects of stress during early development, and exposure to multiple traumatic early life events dramatically increases the risk for AUDs. However, not everyone exposed to early life stress will develop an AUD. The mechanisms determining whether an individual’s brain adapts and becomes resilient to the effects of stress or succumbs and is unable to cope with stress remain elusive. Emerging evidence suggests that neuroplastic changes in the nucleus accumbens (NAc following early life stress underlie the development of AUDs. This review discusses the impact of early life stress on NAc structure and function, how these changes affect cholinergic signaling within the mesolimbic reward pathway and the role nicotinic acetylcholine receptors (nAChRs play in this process. Understanding the neural pathways and mechanism determining stress resilience or susceptibility will improve our ability to identify individuals susceptible to developing AUDs, formulate cognitive interventions to prevent AUDs in susceptible individuals and to elucidate and enhance potential therapeutic targets, such as the nAChRs, for those struggling to overcome an AUD.

  16. Rapid synthesis of acetylcholine receptors at neuromuscular junctions.

    Science.gov (United States)

    Ramsay, D A; Drachman, D B; Pestronk, A

    1988-10-11

    The rate of acetylcholine receptor (AChR) degradation in mature, innervated mammalian neuromuscular junctions has recently been shown to be biphasic; up to 20% are rapidly turned over (RTOs; half life less than 1 day) whereas the remainder are lost more slowly ('stable' AChRs; half life 10-12 days). In order to maintain normal junctional receptor density, synthesis and insertion of AChRs should presumably be sufficiently rapid to replace both the RTOs and the stable receptors. We have tested this prediction by blocking pre-existing AChRs in the mouse sternomastoid muscle with alpha-bungarotoxin (alpha-BuTx), and monitoring the subsequent appearance of 'new' junctional AChRs at intervals of 3 h to 20 days by labeling them with 125I-alpha-BuTx. The results show that new receptors were initially inserted rapidly (16% at 24 h and 28% at 48 h). The rate of increase of 'new' 125I-alpha-BuTx binding sites gradually slowed down during the remainder of the time period studied. Control observations excluded possible artifacts of the experimental procedure including incomplete blockade of AChRs, dissociation of toxin-receptor complexes, or experimentally induced alteration of receptor synthesis. The present demonstration of rapid synthesis and incorporation of AChRs at innervated neuromuscular junctions provides support for the concept of a subpopulation of rapidly turned over AChRs. The RTOs may serve as precursors for the larger population of stable receptors and have an important role in the metabolism of the neuromuscular synapse.

  17. Acetylcholine receptors in dementia and mild cognitive impairment

    Energy Technology Data Exchange (ETDEWEB)

    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.)

  18. The formation of acetylcholine receptor clusters visualized with quantum dots

    Directory of Open Access Journals (Sweden)

    Peng H Benjamin

    2009-07-01

    Full Text Available Abstract Background Motor innervation of skeletal muscle leads to the assembly of acetylcholine receptor (AChR clusters in the postsynaptic membrane at the vertebrate neuromuscular junction (NMJ. Synaptic AChR aggregation, according to the diffusion-mediated trapping hypothesis, involves the establishment of a postsynaptic scaffold that "traps" freely diffusing receptors into forming high-density clusters. Although this hypothesis is widely cited to explain the formation of postsynaptic AChR clusters, direct evidence at molecular level is lacking. Results Using quantum dots (QDs and live cell imaging, we provide new measurements supporting the diffusion-trap hypothesis as applied to AChR cluster formation. Consistent with published works, experiments on cultured Xenopus myotomal muscle cells revealed that AChRs at clusters that formed spontaneously (pre-patterned clusters, also called hot spots and at those induced by nerve-innervation or by growth factor-coated latex beads were very stable whereas diffuse receptors outside these regions were mobile. Moreover, despite the restriction of AChR movement at sites of synaptogenic stimulation, individual receptors away from these domains continued to exhibit free diffusion, indicating that AChR clustering at NMJ does not involve an active attraction of receptors but is passive and diffusion-driven. Conclusion Single-molecular tracking using QDs has provided direct evidence that the clustering of AChRs in muscle cells in response to synaptogenic stimuli is achieved by two distinct cellular processes: the Brownian motion of receptors in the membrane and their trapping and immobilization at the synaptic specialization. This study also provides a clearer picture of the "trap" that it is not a uniformly sticky area but consists of discrete foci at which AChRs are immobilized.

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

    International Nuclear Information System (INIS)

    Schoenbaechler, R.; Westera, G.; Nan-Horng Lin

    2002-01-01

    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 (K i ) 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 [ 11 C]-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 [ 11 C]-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 [ 11 C]-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 [ 11 C]-A-186253.1 in the brain corresponds to specifically bound radioligand, but not to the α4β2 subtype of the nicotinic receptor. (author)

  20. Loss of Acetylcholine Signaling Reduces Cell Clearance Deficiencies in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Sérgio M Pinto

    Full Text Available The ability to eliminate undesired cells by apoptosis is a key mechanism to maintain organismal health and homeostasis. Failure to clear apoptotic cells efficiently can cause autoimmune diseases in mammals. Genetic studies in Caenorhabditis elegans have greatly helped to decipher the regulation of apoptotic cell clearance. In this study, we show that the loss of levamisole-sensitive acetylcholine receptor, but not of a typical neuronal acetylcholine receptor causes a reduction in the number of persistent cell corpses in worms suffering from an engulfment deficiency. This reduction is not caused by impaired or delayed cell death but rather by a partial restoration of the cell clearance capacity. Mutants in acetylcholine turn-over elicit a similar phenotype, implying that acetylcholine signaling is the process responsible for these observations. Surprisingly, tissue specific RNAi suggests that UNC-38, a major component of the levamisole-sensitive receptor, functions in the dying germ cell to influence engulfment efficiency. Animals with loss of acetylcholine receptor exhibit a higher fraction of cell corpses positive for the "eat-me" signal phosphatidylserine. Our results suggest that modulation by ion channels of ion flow across plasma membrane in dying cells can influence the dynamics of phosphatidylserine exposure and thus clearance efficiency.

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

    DEFF Research Database (Denmark)

    Collin, Caitlin Alexis; Hauser, Frank; Gonzalez de Valdivia, Ernesto I

    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 this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M......) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked...

  2. Non-Neuronal Functions of the M2 Muscarinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2013-04-01

    Full Text Available Acetylcholine is an important neurotransmitter whose effects are mediated by two classes of receptors. The nicotinic acetylcholine receptors are ion channels, whereas the muscarinic receptors belong to the large family of G protein coupled seven transmembrane helix receptors. Beyond its function in neuronal systems, it has become evident that acetylcholine also plays an important role in non-neuronal cells such as epithelial and immune cells. Furthermore, many cell types in the periphery are capable of synthesizing acetylcholine and express at least some of the receptors. In this review, we summarize the non-neuronal functions of the muscarinic acetylcholine receptors, especially those of the M2 muscarinic receptor in epithelial cells. We will review the mechanisms of signaling by the M2 receptor but also the cellular trafficking and ARF6 mediated endocytosis of this receptor, which play an important role in the regulation of signaling events. In addition, we provide an overview of the M2 receptor in human pathological conditions such as autoimmune diseases and cancer.

  3. The novel protein kinase C epsilon isoform modulates acetylcholine release in the rat neuromuscular junction.

    Science.gov (United States)

    Obis, Teresa; Hurtado, Erica; Nadal, Laura; Tomàs, Marta; Priego, Mercedes; Simon, Anna; Garcia, Neus; Santafe, Manel M; Lanuza, Maria A; Tomàs, Josep

    2015-12-01

    Various protein kinase C (PKC) isoforms contribute to the phosphorylating activity that modulates neurotransmitter release. In previous studies we showed that nPKCε is confined in the presynaptic site of the neuromuscular junction and its presynaptic function is activity-dependent. Furthermore, nPKCε regulates phorbol ester-induced acetylcholine release potentiation, which further indicates that nPKCε is involved in neurotransmission. The present study is designed to examine the nPKCε involvement in transmitter release at the neuromuscular junction. We use the specific nPKCε translocation inhibitor peptide εV1-2 and electrophysiological experiments to investigate the involvement of this isoform in acetylcholine release. We observed that nPKCε membrane translocation is key to the synaptic potentiation of NMJ, being involved in several conditions that upregulate PKC isoforms coupling to acetylcholine (ACh) release (incubation with high Ca(2+), stimulation with phorbol esters and protein kinase A, stimulation with adenosine 3',5'-cyclic monophosphorothioate, 8-Bromo-, Rp-isomer, sodium salt -Sp-8-BrcAMP-). In all these conditions, preincubation with the nPKCε translocation inhibitor peptide (εV1-2) impairs PKC coupling to acetylcholine release potentiation. In addition, the inhibition of nPKCε translocation and therefore its activity impedes that presynaptic muscarinic autoreceptors and adenosine autoreceptors modulate transmitter secretion. Together, these results point to the importance of nPKCε isoform in the control of acetylcholine release in the neuromuscular junction.

  4. Inhibition of synaptically evoked cortical acetylcholine release by adenosine: an in vivo microdialysis study in the rat.

    Science.gov (United States)

    Materi, L M; Rasmusson, D D; Semba, K

    2000-01-01

    The release of cortical acetylcholine from the intracortical axonal terminals of cholinergic basal forebrain neurons is closely associated with electroencephalographic activity. One factor which may act to reduce cortical acetylcholine release and promote sleep is adenosine. Using in vivo microdialysis, we examined the effect of adenosine and selective adenosine receptor agonists and antagonists on cortical acetylcholine release evoked by electrical stimulation of the pedunculopontine tegmental nucleus in urethane anesthetized rats. All drugs were administered locally within the cortex by reverse dialysis. None of the drugs tested altered basal release of acetylcholine in the cortex. Adenosine significantly reduced evoked cortical acetylcholine efflux in a concentration-dependent manner. This was mimicked by the adenosine A(1) receptor selective agonist N(6)-cyclopentyladenosine and blocked by the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The A(2A) receptor agonist 2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne hydrochloride (CGS 21680) did not alter evoked cortical acetylcholine release even in the presence of DPCPX. Administered alone, neither DPCPX nor the non-selective adenosine receptor antagonist caffeine affected evoked cortical acetylcholine efflux. Simultaneous delivery of the adenosine uptake inhibitors dipyridamole and S-(4-nitrobenzyl)-6-thioinosine significantly reduced evoked cortical acetylcholine release, and this effect was blocked by the simultaneous administration of caffeine. These data indicate that activation of the A(1) adenosine receptor inhibits acetylcholine release in the cortex in vivo while the A(2A) receptor does not influence acetylcholine efflux. Such inhibition of cortical acetylcholine release by adenosine may contribute to an increased propensity to sleep during prolonged wakefulness.

  5. Prejunctional inhibition of norepinephrine release caused by acetylcholine in the human saphenous vein

    International Nuclear Information System (INIS)

    Rorie, D.K.; Rusch, N.J.; Shepherd, J.T.; Vanhoutte, P.M.; Tyce, G.M.

    1981-01-01

    We performed experiments to determine whether or not acetylcholine exerts a prejunctional inhibitory effect on adrenergic neurotransmission in the human blood vessel wall. Rings of human greater saphenous veins were prepared 2 to 15 hours after death and mounted for isometric tension recording in organ chambers filled with Krebs-Ringer solution. Acetylcholine depressed contractile responses to electric activation of the sympathetic nerve endings significantly more than those to exogenous norepinephrine; the relaxations caused by the cholinergic transmitter were antagonized by atropine. Helical strips were incubated with [/sub 3/H]norepinephrine and mounted for superfusion. Electric stimulation augmented the fractional release of labeled norepinephrine. Acetylcholine caused a depression of the evoked /sub 3/H release which was antagonized by atropine but not by hexamethonium. These experiments demonstrate that, as in animal cutaneous veins, there are prejunctional inhibitory muscarinic receptors on the adrenergic nerve endings in the human saphenous vein. By contrast, the human vein also contains postjunctional inhibitory muscarinic receptors

  6. Covalent attachment of antagonists to the a7 nicotinic acetylcholine receptor: synthesis and reactivity of substituted maleimides

    DEFF Research Database (Denmark)

    Ambrus, Joseph I; Halliday, Jill I; Kanizaj, Nicholas

    2012-01-01

    The 3-methylmaleimide congeners of the natural product methyllycaconitine (MLA) and an analogue covalently attach to functional cysteine mutants of the a7 nicotinic acetylcholine receptor (nAChR).......The 3-methylmaleimide congeners of the natural product methyllycaconitine (MLA) and an analogue covalently attach to functional cysteine mutants of the a7 nicotinic acetylcholine receptor (nAChR)....

  7. Involvement of spinal serotonin receptors in the regulation of intraspinal acetylcholine release.

    Science.gov (United States)

    Kommalage, Mahinda; Höglund, A Urban

    2005-02-21

    Stimulation of spinal serotonin (5-HT) receptors results in analgesia and release of acetylcholine. We investigated the involvement of 5-HT1, 5-HT2, and 5-HT3 receptor subtypes in the regulation of spinal acetylcholine release. A spinal microdialysis probe was placed dorsally at about the C5 level in anaesthetized rats. The selective serotonin reuptake inhibitor citalopram was found to increase acetylcholine release when infused via the microdialysis probe. Several doses of the 5-HT receptor agonists 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT, 5-HT1A), 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP93129, 5-HT1B), alpha-methyl-5-hydroxytryptamine maleate (m5-HT, 5-HT2), 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI, 5-HT2C), and 1-(m-chlorophenyl)-biguanide (5-HT3) were subsequently infused via the microdialysis probe. Only 8-OH-DPAT, CP93129, and m5-HT increased acetylcholine release dose dependently. The 5-HT1A receptor selective antagonist (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide hydrochloride and the 5-HT2A receptor selective antagonist ketanserin tartrate inhibited the 8-OH-DPAT and the m5-HT induced acetylcholine release. The results suggest that 5-HT1A and the 5-HT2A receptors are involved in the regulation of acetylcholine release in the spinal cord.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [ 3 H]acetylcholine release from rabbit retina labeled in vitro with [ 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 [ 3 H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [ 3 H]acetylcholine with the following order of potency: apomorphine ≤ SKF(R)82526 3 H]acetylcholine: SCH 23390 (IC50 = 1 nM) 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 [ 3 H]SCH 23390, or as determined by adenylate cyclase activity. [ 3 H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [ 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 [ 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 [ 3 H]SCH 23390 binding sites (r = 0.755, P < .05, n = 8)

  9. Comparative study of muscarinic acetylcholine receptors of human and rat cortical glial cells

    International Nuclear Information System (INIS)

    Demushkin, V.P.; Burbaeva, G.S.; Dzhaliashvili, T.A.; Plyashkevich, Y.G.

    1985-01-01

    The aim of the present investigation was a comparative studyof muscarinic acetylcholine receptors in human and rat glial cells. ( 3 H)Quinuclidinyl-benzylate (( 3 H)-QB), atropine, platiphylline, decamethonium, carbamylcholine, tubocurarine, and nicotine were used. The glial cell fraction was obtained from the cerebral cortex of rats weighing 130-140 g and from the frontal pole of the postmortem brain from men aged 60-70 years. The use of the method of radioimmune binding of ( 3 H)-QB with human and rat glial cell membranes demonstrated the presence of a muscarinic acetylcholine receptor in the glial cells

  10. Orthosteric and allosteric potentiation of heteromeric neuronal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Wang, Jingyi; Lindstrom, Jon

    2018-06-01

    Heteromeric nicotinic ACh receptors (nAChRs) were thought to have two orthodox agonist-binding sites at two α/β subunit interfaces. Highly selective ligands are hard to develop by targeting orthodox agonist sites because of high sequence similarity of this binding pocket among different subunits. Recently, unorthodox ACh-binding sites have been discovered at some α/α and β/α subunit interfaces, such as α4/α4, α5/α4 and β3/α4. Targeting unorthodox sites may yield subtype-selective ligands, such as those for (α4β2) 2 α5, (α4β2) 2 β3 and (α6β2) 2 β3 nAChRs. The unorthodox sites have unique pharmacology. Agonist binding at one unorthodox site is not sufficient to activate nAChRs, but it increases activation from the orthodox sites. NS9283, a selective agonist for the unorthodox α4/α4 site, was initially thought to be a positive allosteric modulator (PAM). NS9283 activates nAChRs with three engineered α4/α4 sites. PAMs, on the other hand, act at allosteric sites where ACh cannot bind. Known PAM sites include the ACh-homologous non-canonical site (e.g. morantel at β/α), the C-terminus (e.g. Br-PBTC and 17β-estradiol), a transmembrane domain (e.g. LY2087101) or extracellular and transmembrane domain interfaces (e.g. NS206). Some of these PAMs, such as Br-PBTC and 17β-estradiol, require only one subunit to potentiate activation of nAChRs. In this review, we will discuss differences between activation from orthosteric and allosteric sites, their selective ligands and clinical implications. These studies have advanced understanding of the structure, assembly and pharmacology of heteromeric neuronal nAChRs. This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc. © 2017 The British Pharmacological Society.

  11. Central nervous system promotes thermotolerance via FoxO/DAF-16 activation through octopamine and acetylcholine signaling in Caenorhabditis elegans.

    Science.gov (United States)

    Furuhashi, Tsubasa; Sakamoto, Kazuichi

    2016-03-25

    The autonomic nervous system (ANS) responds to many kinds of stressors to maintain homeostasis. Although the ANS is believed to regulate stress tolerance, the exact mechanism underlying this is not well understood. To understand this, we focused on longevity genes, which have functions such as lifespan extension and promotion of stress tolerance. To understand the relationship between ANS and longevity genes, we analyzed stress tolerance of Caenorhabditis elegans treated with octopamine, which has an affinity to noradrenaline in insects, and acetylcholine. Octopamine and acetylcholine did not show resistance against H2O2, but the neurotransmitters promoted thermotolerance via DAF-16. However, chronic treatment with octopamine and acetylcholine did not extend the lifespan, although DAF-16 plays an important role in longevity. In conclusion, our results show that octopamine and acetylcholine activate DAF-16 in response to stress, but chronic induction of octopamine and acetylcholine is not beneficial for increasing longevity. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Design, synthesis and biological evaluation of Erythrina alkaloid analogues as neuronal nicotinic acetylcholine receptor antagonists

    DEFF Research Database (Denmark)

    Crestey, François; Jensen, Anders A.; Borch, Morten

    2013-01-01

    The synthesis of a new series of Erythrina alkaloid analogues and their pharmacological characterization at various nicotine acetylcholine receptor (nAChR) subtypes are described. The compounds were designed to be simplified analogues of aromatic erythrinanes with the aim of obtaining subtype...

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

    DEFF Research Database (Denmark)

    Petersen, Ida Nymann; Crestey, François; Jensen, Anders A

    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...

  15. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition

    DEFF Research Database (Denmark)

    Kachel, Hamid S; Patel, Rohit N; Franzyk, Henrik

    2016-01-01

    -fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents...

  16. Functional aspects of dexamethasone upregulated nicotinic acetylcholine receptors in C2C12 myotubes

    NARCIS (Netherlands)

    Maestrone, E; Lagostena, L; Henning, RH; DenHertog, A; Nobile, M

    Three days of treatment with the glucocorticoid dexamethasone (1 nM-mu M) induced a concentration-dependent up-regulation of muscle nicotinic acetylcholine receptor (nAChR) in C2C12 mouse myotubes (EC(50)=10+/-7.3 nM), as assessed by [H-3]alpha-BuTx binding. The maximum increase in binding amounted

  17. Intersubunit bridge formation governs agonist efficacy at nicotinic acetylcholine alpha4beta2 receptors

    DEFF Research Database (Denmark)

    Rohde, Line Aagot Hede; Ahring, Philip Kiær; Jensen, Marianne Lerbech

    2012-01-01

    The a4ß2 subtype of the nicotinic acetylcholine receptor (nAChR) has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Still, a thorough understanding of structure-function relationships of a4ß2 agonists is lacking...

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

    DEFF Research Database (Denmark)

    Jepsen, Tue H.; Jensen, Anders A.; Lund, Mads Henrik

    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...

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

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Christensen, Ditte Z; Hansen, Henrik H

    2009-01-01

    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 induced by PCP...

  20. Cognitive improvement by activation of alpha7 nicotinic acetylcholine receptors: from animal models to human pathophysiology

    DEFF Research Database (Denmark)

    Thomsen, Morten S; Hansen, Henrik H; Timmerman, Daniel B

    2010-01-01

    Agonists and positive allosteric modulators of the alpha(7) nicotinic acetylcholine receptor (nAChR) are currently being developed for the treatment of cognitive disturbances in patients with schizophrenia or Alzheimer's disease. This review describes the neurobiological properties of the alpha n...

  1. Nicotinic Acetylcholine Receptor Expression and Susceptibility to Cholinergic Immunomodulation in Human Monocytes of Smoking Individuals

    NARCIS (Netherlands)

    van der Zanden, Esmerij P.; Hilbers, Francisca W.; Verseijden, Caroline; van den Wijngaard, Rene M.; Skynner, Mike; Lee, Kevin; Ulloa, Luis; Boeckxstaens, Guy E.; de Jonge, Wouter J.

    2012-01-01

    Objective: Smoking is generally accepted as a factor that affects the disease course in inflammatory bowel disease patients. Whether these effects can be contributed to the immunomodulatory effects of nicotine via nicotinic acetylcholine receptor (nAChR) activation is unclear. As previous data

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

    DEFF Research Database (Denmark)

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

    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...

  3. Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

    Directory of Open Access Journals (Sweden)

    Ann Sluder

    Full Text Available The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

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

    NARCIS (Netherlands)

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

    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

  5. Effect of basal forebrain stimulation on extracellular acetylcholine release and blood flow in the olfactory bulb.

    Science.gov (United States)

    Uchida, Sae; Kagitani, Fusako

    2017-05-12

    The olfactory bulb receives cholinergic basal forebrain input, as does the neocortex; however, the in vivo physiological functions regarding the release of extracellular acetylcholine and regulation of regional blood flow in the olfactory bulb are unclear. We used in vivo microdialysis to measure the extracellular acetylcholine levels in the olfactory bulb of urethane-anesthetized rats. Focal chemical stimulation by microinjection of L-glutamate into the horizontal limb of the diagonal band of Broca (HDB) in the basal forebrain, which is the main source of cholinergic input to the olfactory bulb, increased extracellular acetylcholine release in the ipsilateral olfactory bulb. When the regional cerebral blood flow was measured using laser speckle contrast imaging, the focal chemical stimulation of the HDB did not significantly alter the blood flow in the olfactory bulb, while increases were observed in the neocortex. Our results suggest a functional difference between the olfactory bulb and neocortex regarding cerebral blood flow regulation through the release of acetylcholine by cholinergic basal forebrain input.

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

    Czech Academy of Sciences Publication Activity Database

    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

  7. Variants in SLC18A3, vesicular acetylcholine transporter, cause congenital myasthenic syndrome

    NARCIS (Netherlands)

    O'Grady, Gina L.; Verschuuren, Corien; Yuen, Michaela; Webster, Richard; Menezes, Manoj; Fock, Johanna M.; Pride, Natalie; Best, Heather A.; Damm, Tatiana Benavides; Turner, Christian; Lek, Monkol; Engel, Andrew G.; North, Kathryn N.; Clarke, Nigel F.; MacArthur, Daniel G.; Kamsteeg, Erik-Jan; Cooper, Sandra T.

    2016-01-01

    Objective: To describe the clinical and genetic characteristics of presynaptic congenital myasthenic syndrome secondary to biallelic variants in SLC18A3. Methods: Individuals from 2 families were identified with biallelic variants in SLC18A3, the gene encoding the vesicular acetylcholine transporter

  8. Cloning and structural analysis of partial acetylcholine receptor subunit genes from the parasitic nematode Teladorsagia circumcincta

    NARCIS (Netherlands)

    Walker, J.; Hoekstra, R.; Roos, M. H.; Wiley, L. J.; Weiss, A. S.; Sangster, N. C.; Tait, A.

    2001-01-01

    Nematode nicotinic acetylcholine receptors (nAChRs) are the sites of action for the anthelmintic drug levamisole. Recent findings indicate that the molecular mechanism of levamisole resistance may involve changes in the number and/or functions of target nAChRs. Accordingly, we have used an RT-PCR

  9. Characterization of the positive and negative inotropic effects of acetylcholine in the human myocardium

    NARCIS (Netherlands)

    X.Y. Du (Xiaoyi); R.G. Schoemaker (Regien); E. Bos (Egbert); P.R. Saxena (Pramod Ranjan)

    1995-01-01

    textabstractIn the human isolated myocardium, acetylcholine (10−9 to 10−3 M) elicited a biphasic inotropic effect (a decrease in the lower and an increase in the higher concentration range) in atrial and a positive inotropic effect in ventricular trabeculae. However, under conditions of raised

  10. Layer-specific modulation of the prefrontal cortex by nicotinic acetylcholine receptors

    NARCIS (Netherlands)

    Poorthuis, R.B.; Bloem, B.; Schak, B.; Wester, J.; de Kock, C.P.J.; Mansvelder, H.D.

    2013-01-01

    Acetylcholine signaling through nicotinic receptors (nAChRs) in the prefrontal cortex (PFC) is crucial for attention. Nicotinic AChRs are expressed on glutamatergic inputs to layer V (LV) cells and on LV interneurons and LVI pyramidal neurons. Whether PFC layers are activated by nAChRs to a similar

  11. Weak toxin WTX from Naja kaouthia cobra venom interacts with both nicotinic and muscarinic acetylcholine receptors

    Czech Academy of Sciences Publication Activity Database

    Mordvintsev, D.Y.; Polyak, Y.L.; Rodionov, D.I.; Jakubík, Jan; Doležal, Vladimír; Karlsson, E.; Tsetlin, V.I.; Utkin, Y.N.

    2009-01-01

    Roč. 276, č. 18 (2009), s. 5065-5075 ISSN 1742-464X R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA500110703 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine receptors * allosteric interaction * toxins Subject RIV: ED - Physiology Impact factor: 3.042, year: 2009

  12. Muscarinic Acetylcholine Receptors Act in Synergy to Facilitate Learning and Memory

    Science.gov (United States)

    Leaderbrand, Katherine; Chen, Helen J.; Corcoran, Kevin A.; Guedea, Anita L.; Jovasevic, Vladimir; Wess, Jurgen; Radulovic, Jelena

    2016-01-01

    Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on…

  13. Apolipoprotein E4 reduces evoked hippocampal acetylcholine release in adult mice

    Czech Academy of Sciences Publication Activity Database

    Dolejší, Eva; Liraz, O.; Rudajev, Vladimír; Zimčík, Pavel; Doležal, Vladimír; Michaelson, D. M.

    2016-01-01

    Roč. 136, č. 3 (2016), s. 503-509 ISSN 0022-3042 R&D Projects: GA MŠk(CZ) LH13269 Institutional support: RVO:67985823 Keywords : acetylcholine release * Alzheimer's disease (AD) * apolipoprotein E4 (apoE4) * hippocampus Subject RIV: FH - Neurology Impact factor: 4.083, year: 2016

  14. Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors

    Czech Academy of Sciences Publication Activity Database

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

    2017-01-01

    Roč. 7, Jan 16 (2017), č. článku 40381. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GBP304/12/G069 Institutional support: RVO:67985823 Keywords : muscarinic acetylcholine receptors * N-methylscopolamine * ligand binding * molecular dynamics Subject RIV: ED - Physiology OBOR OECD: Physiology (including cytology) Impact factor: 4.259, year: 2016

  15. Fragment growing induces conformational changes in acetylcholine-binding protein: A structural and thermodynamic analysis

    NARCIS (Netherlands)

    Edink, E.S.; Rucktooa, P.; Retra, K.; Akdemir, A.; Nahar, T.T.; Zuiderveld, O.P.; van Elk, R.; Janssen, E.; van Nierop, P.; van Muijlwijk-Koezen, J.E.; Smit, A.B.; Sixma, T.K.; Leurs, R.; de Esch, I.J.P.

    2011-01-01

    Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP).

  16. Nicotinic acetylcholine receptor β2-subunits in the medial prefrontal cortex control attention

    NARCIS (Netherlands)

    Guillem, K.; Bloem, B.; Poorthuis, R.B.; Loos, M.; Smit, A.B.; Maskos, U.; Spijker, S.; Mansvelder, H.D.

    2011-01-01

    More than one-third of all people are estimated to experience mild to severe cognitive impairment as they age. Acetylcholine (ACh) levels in the brain diminish with aging, and nicotinic ACh receptor (nAChR) stimulation is known to enhance cognitive performance. The prefrontal cortex (PFC) is

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

    Science.gov (United States)

    Courtot, Elise; Charvet, Claude L.; Beech, Robin N.; Harmache, Abdallah; Wolstenholme, Adrian J.; Holden-Dye, Lindy; O’Connor, Vincent; Peineau, Nicolas; Woods, Debra J.; Neveu, Cedric

    2015-01-01

    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. PMID:26625142

  18. Bradykinin or acetylcholine as vasodilators to test endothelial venous function in healthy subjects

    Directory of Open Access Journals (Sweden)

    Eneida R. Rabelo

    2008-01-01

    Full Text Available INTRODUCTION: The evaluation of endothelial function has been performed in the arterial bed, but recently evaluation within the venous system has also been explored. Endothelial function studies employ different drugs that act as endothelium-dependent vasodilatory response inductors. OBJECTIVES: The aim of this study is to compare the endothelium-dependent venous vasodilator response mediated by either acetylcholine or bradykinin in healthy volunteers. METHODS AND RESULTS: Changes in vein diameter after phenylephrine-induced venoconstriction were measured to compare venodilation induced by acetylcholine or bradykinin (linear variable differential transformer dorsal hand vein technique. We studied 23 healthy volunteers; 31% were male, and the subject had a mean age of 33 ± 8 years and a mean body mass index of 23 ± 2 kg/m². The maximum endothelium-dependent venodilation was similar for both drugs (p = 0.13, as well as the mean responses for each dose of both drugs (r = 0.96. The maximum responses to acetylcholine and bradykinin also had good agreement. CONCLUSION: There were no differences between acetylcholine and bradykinin as venodilators in this endothelial venous function investigation.

  19. GABAergic Neurons of the Rat Dorsal Hippocampus Express Muscarinic Acetylcholine Receptors

    NARCIS (Netherlands)

    van der Zee, E.A.; Luiten, P.G.M.

    1993-01-01

    The expression of muscarinic acetylcholine receptors (mAChRs) in glutamic acid decarboxylase (GAD)-positive cells in the different strata of CA1, CA3, and the dentate gyrus (DG) of the dorsal hippocampus is examined by way of quantitative immunofluorescent double labeling employing M35, the

  20. Effects of acute chlorpyrifos exposure on in vivo acetylcholine accumulation in rat striatum

    International Nuclear Information System (INIS)

    Karanth, Subramanya; Liu, Jing; Mirajkar, Nikita; Pope, Carey

    2006-01-01

    This study examined the acute effects of chlorpyrifos (CPF) on cholinesterase inhibition and acetylcholine levels in the striatum of freely moving rats using in vivo microdialysis. Adult, male Sprague-Dawley rats were treated with vehicle (peanut oil, 2 ml/kg) or CPF (84, 156 or 279 mg/kg, sc) and functional signs of toxicity, body weight and motor activity recorded. Microdialysis was conducted at 1, 4 and 7 days after CPF exposure for measurement of acetylcholine levels in striatum. Rats were then sacrificed and the contralateral striatum and diaphragm were collected for biochemical measurements. Few overt signs of cholinergic toxicity were noted in any rats. Body weight gain was significantly affected in the high-dose (279 mg/kg) group only, while motor activity (nocturnal rearing) was significantly reduced in all CPF-treated groups at one day (84 mg/kg) or from 1-4 days (156 and 279 mg/kg) after dosing. Cholinesterase activities in both diaphragm and striatum were markedly inhibited (50-92%) in a time-dependent manner, but there were relatively minimal dose-related changes. In contrast, time- and dose-dependent changes in striatal acetylcholine levels were noted, with significantly higher levels noted in the high-dose group compared to other groups. Maximal increases in striatal acetylcholine levels were observed at 4-7 days after dosing (84 mg/kg, 7-9-fold; 156 mg/kg, 10-13-fold; 279 mg/kg, 35-57-fold). Substantially higher acetylcholine levels were noted when an exogenous cholinesterase inhibitor was included in the perfusion buffer, but CPF treatment-related differences were substantially lower in magnitude under those conditions. The results suggest that marked differences in acetylcholine accumulation can occur with dosages of CPF eliciting relatively similar degrees of cholinesterase inhibition. Furthermore, the minimal expression of classic signs of cholinergic toxicity in the presence of extensive brain acetylcholine accumulation suggests that some

  1. Topical Non-Iontophoretic Application of Acetylcholine and Nitroglycerin via a Translucent Patch: A New Means for Assessing Microvascular Reactivity

    Science.gov (United States)

    Schonberger, Robert B.; Worden, William S.; Shahmohammadi, Kaveh; Menn, Kirsten; Silverman, Tyler J.; Stout, Robert G.; Shelley, Kirk H.; Silverman, David G.

    2007-01-01

    Objective: Assessments of endothelial cell function with acetylcholine have typically used systemic, regional intra-arterial, or iontophoretic delivery of drug. Each of these techniques induces systemic and/or local changes that compromise their safety or effectiveness. Using translucent drug preparations applied under laser Doppler flowmetry (LDF) probes, we tested whether local vasodilation can be induced with non-iontophoretic transdermal delivery of acetylcholine and how such dilation would compare to the dilation achieved with topical nitroglycerin in healthy volunteers. Methods: Ten subjects without known vascular disease were recruited for LDF monitoring at sites of drug application for this preliminary investigation. Topical acetylcholine chloride, nitroglycerin, and placebo were applied via translucent patches to the forehead directly below LDF probes. Results: LDF readings increased by 406 percent (245 percent to 566 percent) and 36 percent (26 percent to 46 percent), respectively, at the acetylcholine and placebo sites (p = .005 by Wilcoxon Signed Rank Test (WSRT) for acetylcholine vs. placebo); and they increased by 365 percent (179 percent to 550 percent) at the nitroglycerin site (p = .005 by WSRT for nitroglycerin vs. placebo; p = .6 vs. acetylcholine). Conclusion: Transdermal delivery of acetylcholine can induce significant local vasodilatory responses comparable to those achieved with nitroglycerin without requiring iontophoresis. The means of transdermal delivery and monitoring described herein may constitute a new minimally invasive way to interrogate the microvasculature and thereby assess the microcirculatory changes induced by various disorders and therapeutic interventions. PMID:17876370

  2. Concomitant release of ventral tegmental acetylcholine and accumbal dopamine by ghrelin in rats.

    Directory of Open Access Journals (Sweden)

    Elisabet Jerlhag

    Full Text Available Ghrelin, an orexigenic peptide, regulates energy balance specifically via hypothalamic circuits. Growing evidence suggest that ghrelin increases the incentive value of motivated behaviours via activation of the cholinergic-dopaminergic reward link. It encompasses the cholinergic afferent projection from the laterodorsal tegmental area (LDTg to the dopaminergic cells of the ventral tegmental area (VTA and the mesolimbic dopamine system projecting from the VTA to nucleus accumbens (N.Acc.. Ghrelin receptors (GHS-R1A are expressed in these reward nodes and ghrelin administration into the LDTg increases accumbal dopamine, an effect involving nicotinic acetylcholine receptors in the VTA. The present series of experiments were undertaken directly to test this hypothesis. Here we show that ghrelin, administered peripherally or locally into the LDTg concomitantly increases ventral tegmental acetylcholine as well as accumbal dopamine release. A GHS-R1A antagonist blocks this synchronous neurotransmitter release induced by peripheral ghrelin. In addition, local perfusion of the unselective nicotinic antagonist mecamylamine into the VTA blocks the ability of ghrelin (administered into the LDTg to increase N.Acc.-dopamine, but not VTA-acetylcholine. Collectively our data indicate that ghrelin activates the LDTg causing a release of acetylcholine in the VTA, which in turn activates local nicotinic acetylcholine receptors causing a release of accumbal dopamine. Given that a dysfunction in the cholinergic-dopaminergic reward system is involved in addictive behaviours, including compulsive overeating and alcohol use disorder, and that hyperghrelinemia is associated with such addictive behaviours, ghrelin-responsive circuits may serve as a novel pharmacological target for treatment of alcohol use disorder as well as binge eating.

  3. Recent Duplication and Functional Divergence in Parasitic Nematode Levamisole-Sensitive Acetylcholine Receptors.

    Directory of Open Access Journals (Sweden)

    Thomas B Duguet

    2016-07-01

    Full Text Available Helminth parasites rely on fast-synaptic transmission in their neuromusculature to experience the outside world and respond to it. Acetylcholine plays a pivotal role in this and its receptors are targeted by a wide variety of both natural and synthetic compounds used in human health and for the control of parasitic disease. The model, Caenorhabditis elegans is characterized by a large number of acetylcholine receptor subunit genes, a feature shared across the nematodes. This dynamic family is characterized by both gene duplication and loss between species. The pentameric levamisole-sensitive acetylcholine receptor has been characterized from C. elegans, comprised of five different subunits. More recently, cognate receptors have been reconstituted from multiple parasitic nematodes that are found to vary in subunit composition. In order to understand the implications of receptor composition change and the origins of potentially novel drug targets, we investigated a specific example of subunit duplication based on analysis of genome data for 25 species from the 50 helminth genome initiative. We found multiple independent duplications of the unc-29, acetylcholine receptor subunit, where codon substitution rate analysis identified positive, directional selection acting on amino acid positions associated with subunit assembly. Characterization of four gene copies from a model parasitic nematode, Haemonchus contortus, demonstrated that each copy has acquired unique functional characteristics based on phenotype rescue of transgenic C. elegans and electrophysiology of receptors reconstituted in Xenopus oocytes. We found evidence that a specific incompatibility has evolved for two subunits co-expressed in muscle. We demonstrated that functional divergence of acetylcholine receptors, driven by directional selection, can occur more rapidly than previously thought and may be mediated by alteration of receptor assembly. This phenomenon is common among the

  4. Sigma receptor ligand N,N'-di-(ortho-tolyl)guanidine inhibits release of acetylcholine in the guinea pig ileum.

    Science.gov (United States)

    Cambell, B G; Keana, J F; Weber, E

    1991-11-26

    The inhibition of stimulated contractions of the guinea pig ileum longitudinal muscle/myenteric plexus preparation by sigma receptor ligands has been previously described. In this study, the stimulated release of [3H]acetylcholine from cholinergic nerve terminals in this same preparation was monitored in the presence and absence of sigma receptor ligands. N,N'-Di-(orthotolyl)guanidine (DTG) and other compounds selective for the sigma receptor inhibited stimulated [3H]acetylcholine release. These results suggest that their inhibition of stimulated contractions in this preparation was mediated by inhibition of acetylcholine release.

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

    DEFF Research Database (Denmark)

    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...

  6. Acetylcholine produces contraction mediated by cyclooxigenase pathway in arterial vessels in the marine fish (Isacia conceptionis

    Directory of Open Access Journals (Sweden)

    FA. Moraga

    Full Text Available Preliminary studies showed that dorsal artery contraction mediated by acetylcholine (ACh is blocked with indomethacin in intertidal fish (G. laevifrons. Our objective was to characterize the cholinergic pathway in several artery vessels of the I. conceptionis. Afferent and efferent branchial, dorsal and mesenteric arteries were dissected of 6 juvenile specimens, isometric tension studies were done using doses response curves (DRC for Ach (10–13 to 10–3 M, and cholinergic pathways were obtained by blocking with atropine or indomethacin. CRC to ACh showed a pattern of high sensitivity only in efferente branchial artery and low sensibility in all vessels. Furthermore, these contractions were blocked in the presence of atropine and indomethacin in all vessels. Our results corroborate previous results observed in intertidal species that contraction induced by acetylcholine is mediated by receptors that activate a cyclooxygenase contraction pathway.

  7. Study of the Peripheral Nerve Fibers Myelin Structure Changes during Activation of Schwann Cell Acetylcholine Receptors.

    Directory of Open Access Journals (Sweden)

    Ekaterina E Verdiyan

    Full Text Available In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC acetylcholine receptors (AChRs and the axon excitation (different intervals between action potentials (APs. Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the "axon-SC" interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+-influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization.

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

    International Nuclear Information System (INIS)

    Fatranska, M.; Budai, D.; Gulya, K; Kvetnansky, R.

    1989-01-01

    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 [ 3 H](-) 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. Functional Characterization of CCHamide and Muscarinic Acetylcholine Receptor Signalling in Drosophila melanogaster

    DEFF Research Database (Denmark)

    Ren, Guilin Robin

    G-protein coupled receptors (GPCRs) constitute a large and ancient superfamily of membraneproteins responsible for the transduction of extracellular signals to the inside of the cells. In thisPh.D. thesis, Drosophila melanogaster (Dm) was used as a model organism to investigate a numberof topics...... is a newly discovered insect peptide hormone. The function of this novel peptide hasnot been well characterised. In this Ph.D. thesis, I identified CCHamide-2 peptides in endocrinecells of the gut and neurones of the brain of larvae and endocrine cells of the gut of adultDrosophila. Behavioural assays...... little is known about muscarinic acetylcholine receptorsignalling in insects. In this study, I found that two types of mAChRs occur in D. melanogaster, onecoupling to Gq (A-type) and the other to Gi (B-type). Both A- and B-type Dm-mAChRs can beactivated by acetylcholine (ACh), but the classical...

  10. Uptake of 3H-choline and synthesis of 3H-acetylcholine by human penile corpus cavernosum

    International Nuclear Information System (INIS)

    Blanco, R.; Saenz de Tejada, I.; Azadzoi, K.; Goldstein, I.; Krane, R.J.; Wotiz, H.H.; Cohen, R.A.

    1986-01-01

    The neuroeffectors which relax penile smooth muscle and lead to erection are unknown; physiological studies of human corpus cavernosum, in vitro, have suggested a significant role of cholinergic neurotransmission. To further characterize the importance of cholinergic nerves, biopsies of human corpus cavernosum were obtained at the time of penile prosthesis implantation. Tissues were incubated in 3 H-choline (10 -5 M, 80 Ci/mmol) in oxygenated physiological salt solution at 37 0 C, pH 7.4 for 1 hour. Radiolabelled compounds were extracted with perchloric acid (0.4 M) and acetylcholine and choline were separated by HPLC; 14 C-acetylcholine was used as internal standard. 3 H-choline was accumulated by the tissues (20 +/- 1.9 fmol/mg), and 3 H-acetylcholine was synthesized (4.0 +/- 1.1 fmol/mg). In control experiments, heating of the tissue blocked synthesis of 3 H-acetylcholine. Inhibition of high affinity choline transport by hemicholinium-3 (10 -5 M) diminished tissue accumulation of 3 H-choline and significantly reduced the synthesis of 3 H-acetylcholine (0.5 +/ 0.2 fmol/mg, p < 0.05). These results provide direct evidence of neuronal accumulation of choline and enzymatic conversion to acetylcholine in human corpus cavernosum. Taken together with the physiological studies, it can be concluded that cholinergic neurotransmission in human corpus cavernosum plays a role in penile erection

  11. Fluoxetine Alleviates Behavioral Depression while Decreasing Acetylcholine Release in the Nucleus Accumbens Shell

    OpenAIRE

    Chau, David T; Rada, Pedro V; Kim, Kay; Kosloff, Rebecca A; Hoebel, Bartley G

    2011-01-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have demonstrated the ability to alleviate behavioral depression in the forced swim test; however, the sites and mechanisms of their actions remain to be further elucidated. Previous studies have suggested that behavioral depression in the swim test is mediated in part by acetylcholine (ACh) stimulating the cholinergic M1 receptors in the nucleus accumbens (NAc) shell. The current study tested whether acute, local, and chronic, subc...

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

    Czech Academy of Sciences Publication Activity Database

    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: 4.816, year: 2015

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

    International Nuclear Information System (INIS)

    Raftery, M.A.; Dunn, S.M.J.; Conti-Tronconi, B.M.; Middlemas, D.S.; Crawford, R.D.

    1983-01-01

    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

  14. Nicotine at clinically relevant concentrations affects atrial inward rectifier potassium current sensitive to acetylcholine.

    Science.gov (United States)

    Bébarová, Markéta; Matejovič, Peter; Švecová, Olga; Kula, Roman; Šimurdová, Milena; Šimurda, Jiří

    2017-05-01

    Nicotine abuse is associated with variety of diseases including arrhythmias, most often atrial fibrillation (AF). Altered inward rectifier potassium currents including acetylcholine-sensitive current I K(Ach) are known to be related to AF pathogenesis. Since relevant data are missing, we aimed to investigate I K(Ach) changes at clinically relevant concentrations of nicotine. Experiments were performed by the whole cell patch clamp technique at 23 ± 1 °C on isolated rat atrial myocytes. Nicotine was applied at following concentrations: 4, 40 and 400 nM; ethanol at 20 mM (∼0.09%). Nicotine at 40 and 400 nM significantly activated constitutively active component of I K(Ach) with the maximum effect at 40 nM (an increase by ∼100%); similar effect was observed at -110 and -50 mV. Changes at 4 nM nicotine were negligible on average. Coapplication of 40 nM nicotine and 20 mM ethanol (which is also known to activate this current) did not show cumulative effect. In the case of acetylcholine-induced component of I K(Ach) , a dual effect of nicotine and its correlation with the current magnitude in control were apparent: the current was increased by nicotine in the cells showing small current in control and vice versa. The effect of 40 and 400 nM nicotine on acetylcholine-induced component of I K(Ach) was significantly different at -110 and -50 mV. We conclude that nicotine at clinically relevant concentrations significantly increased constitutively active component of I K(Ach) and showed a dual effect on its acetylcholine-induced component, similarly as ethanol. Synchronous application of nicotine and ethanol did not cause additive effect.

  15. Brain nicotinic acetylcholine receptors are involved in stress-induced potentiation of nicotine reward in rats.

    Science.gov (United States)

    Javadi, Parastoo; Rezayof, Ameneh; Sardari, Maryam; Ghasemzadeh, Zahra

    2017-07-01

    The aim of the present study was to examine the possible role of nicotinic acetylcholine receptors of the dorsal hippocampus (CA1 regions), the medial prefrontal cortex or the basolateral amygdala in the effect of acute or sub-chronic stress on nicotine-induced conditioned place preference. Our results indicated that subcutaneous administration of nicotine (0.2 mg/kg) induced significant conditioned place preference. Exposure to acute or sub-chronic elevated platform stress potentiated the response of an ineffective dose of nicotine. Pre-conditioning intra-CA1 (0.5-4 µg/rat) or intra-medial prefrontal cortex (0.2-0.3 µg/rat) microinjection of mecamylamine (a non-selective nicotinic acetylcholine receptor antagonist) reversed acute stress-induced potentiation of nicotine reward as measured in the conditioned place preference paradigm. By contrast, pre-conditioning intra-basolateral amygdala microinjection of mecamylamine (4 µg/rat) potentiated the effects of acute stress on nicotine reward. Our findings also showed that intra-CA1 or intra-medial prefrontal cortex, but not intra-basolateral amygdala, microinjection of mecamylamine (4 µg/rat) prevented the effect of sub-chronic stress on nicotine reward. These findings suggest that exposure to elevated platform stress potentiates the rewarding effect of nicotine which may be associated with the involvement of nicotinic acetylcholine receptors. It seems that there is a different contribution of the basolateral amygdala, the medial prefrontal cortex or the CA1 nicotinic acetylcholine receptors in stress-induced potentiation of nicotine-induced conditioned place preference.

  16. Autoradiographic visualization of extrajunctional acetylcholine receptors in whole human biceps brachii muscle

    International Nuclear Information System (INIS)

    Askmark, H.; Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    The morphological distribution of acetylcholine receptors in the whole biceps brachii muscle from a patient with amyotrophic lateral sclerosis (ALS) and from a control patient was studied by in vitro autoradiography with 3 H-alpha-bungarotoxin alphaBtx). In ALS, 3 H-alpha-Btx binding occurred over the entire muscle, while in the control case the binding was restricted to the motor end-plate region. (author)

  17. Nerve terminal contributes to acetylcholine receptor organization at the dystrophic neuromuscular junction of mdx mice.

    Science.gov (United States)

    Marques, Maria Julia; Taniguti, Ana Paula Tiemi; Minatel, Elaine; Neto, Humberto Santo

    2007-02-01

    Changes in the distribution of acetylcholine receptors have been reported to occur at the neuromuscular junction of mdx mice and may be a consequence of muscle fiber regeneration rather than the absence of dystrophin. In the present study, we examined whether the nerve terminal determines the fate of acetylcholine receptor distribution in the dystrophic muscle fibers of mdx mice. The left sternomastoid muscle of young (1-month-old) and adult (6-month-old) mdx mice was injected with 60 microl lidocaine hydrochloride to induce muscle degeneration-regeneration. Some mice had their sternomastoid muscle denervated at the time of lidocaine injection. After 10 days of muscle denervation, nerve terminals and acetylcholine receptors were labeled with 4-Di-2-ASP and rhodamine-alpha-bungarotoxin, respectively, for confocal microscopy. In young mdx mice, 75% (n = 137 endplates) of the receptors were distributed in islands. The same was observed in 100% (n = 114 endplates) of the adult junctions. In denervated-regenerated fibers of young mice, the receptors were distributed as branches in 89% of the endplates (n = 90). In denervated-regenerated fibers of adult mice, the receptors were distributed in islands in 100% of the endplates (n = 100). These findings show that nerve-dependent mechanisms are also involved in the changes in receptor distribution in young dystrophic muscles. In older dystrophic muscles, other factors may play a role in receptor distribution.

  18. The anthelmintic levamisole is an allosteric modulator of human neuronal nicotinic acetylcholine receptors.

    Science.gov (United States)

    Levandoski, Mark M; Piket, Barbara; Chang, Jane

    2003-06-13

    L-[-]-2,3,5,6-Tetrahydro-6-phenylimidazo[2,1b]-thiazole hydrochloride (levamisole) is an anthelmintic that targets the nicotinic acetylcholine receptors of parasitic nematodes. We report here the effects of levamisole on human neuronal alpha 3 beta 2 and alpha 3 beta 4 nicotinic receptors, heterologously expressed in Xenopus oocytes and studied with the voltage clamp method. Applied alone, levamisole was a very weak partial agonist for the two subunit combinations. When co-applied with acetylcholine, micromolar concentrations of levamisole potentiated responses, while millimolar concentrations inhibited them; these effects were complex functions of both acetylcholine and levamisole concentrations. The differences in the levamisole effects on the two receptor combinations suggest that the effects are mediated by the beta subunit. Several combinations of agonist and anthelmintic gave the dual potentiation/inhibition behavior, suggesting that the modulatory effects are general. Levamisole inhibition showed macroscopic characteristics of open channel block. Several results led us to conclude that levamisole potentiation occurs through noncompetitive binding to the receptor. We propose pseudo-site binding for noncompetitive potentiation by levamisole.

  19. Monkey Adrenal Chromaffin Cells Express α6β4* Nicotinic Acetylcholine Receptors

    Science.gov (United States)

    Scadden, Mick´l; Carmona-Hidalgo, Beatriz; McIntosh, J. Michael; Albillos, Almudena

    2014-01-01

    Nicotinic acetylcholine receptors (nAChRs) that contain α6 and β4 subunits have been demonstrated functionally in human adrenal chromaffin cells, rat dorsal root ganglion neurons, and on noradrenergic terminals in the hippocampus of adolescent mice. In human adrenal chromaffin cells, α6β4* nAChRs (the asterisk denotes the possible presence of additional subunits) are the predominant subtype whereas in rodents, the predominant nAChR is the α3β4* subtype. Here we present molecular and pharmacological evidence that chromaffin cells from monkey (Macaca mulatta) also express α6β4* receptors. PCR was used to show the presence of transcripts for α6 and β4 subunits and pharmacological characterization was performed using patch-clamp electrophysiology in combination with α-conotoxins that target the α6β4* subtype. Acetylcholine-evoked currents were sensitive to inhibition by BuIA[T5A,P6O] and MII[H9A,L15A]; α-conotoxins that inhibit α6-containing nAChRs. Two additional agonists were used to probe for the expression of α7 and β2-containing nAChRs. Cells with currents evoked by acetylcholine were relatively unresponsive to the α7-selctive agonist choline but responded to the agonist 5-I-A-85380. These studies provide further insights into the properties of natively expressed α6β4* nAChRs. PMID:24727685

  20. Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Satoshi Suo

    Full Text Available Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

  1. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    Science.gov (United States)

    Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

    2017-01-01

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

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

    Science.gov (United States)

    Collin, Caitlin; Hauser, Frank; Gonzalez de Valdivia, Ernesto; de Valdivia, Ernesto Gonzalez; Li, Shizhong; Reisenberger, Julia; Carlsen, Eva M M; Khan, Zaid; Hansen, Niels O; Puhm, Florian; Søndergaard, Leif; Niemiec, Justyna; Heninger, Magdalena; Ren, Guilin R; Grimmelikhuijzen, Cornelis J P

    2013-09-01

    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 this study, we cloned two structurally related GPCRs from the fruit fly Drosophila melanogaster, which, after expression in Chinese hamster ovary cells, proved to be muscarinic acetylcholine receptors. One mAChR (the A-type; encoded by gene CG4356) is activated by acetylcholine (EC50, 5 × 10(-8) M) and muscarine (EC50, 6 × 10(-8) M) and blocked by the classical mAChR antagonists atropine, scopolamine, and 3-quinuclidinyl-benzilate (QNB), while the other (the B-type; encoded by gene CG7918) is also activated by acetylcholine, but has a 1,000-fold lower sensitivity to muscarine, and is not blocked by the antagonists. A- and B-type mAChRs were also cloned and functionally characterized from the red flour beetle Tribolium castaneum. Recently, Haga et al. (Nature 2012, 482: 547-551) published the crystal structure of the human m2 mAChR, revealing 14 amino acid residues forming the binding pocket for QNB. These residues are identical between the human m2 and the D. melanogaster and T. castaneum A-type mAChRs, while many of them are different between the human m2 and the B-type receptors. Using bioinformatics, one orthologue of the A-type and one of the B-type mAChRs could also be found in all other arthropods with a sequenced genome. Protostomes, such as arthropods, and deuterostomes, such as mammals and other vertebrates, belong to two evolutionarily distinct lineages of animal evolution that split about 700 million years ago. We found that animals that originated before this split, such as cnidarians (Hydra), had two A-type mAChRs. From these data we propose a model for the evolution of mAChRs.

  3. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Catherine A Vulfius

    Full Text Available Phospholipases A2 (PLA2s are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Local application of SCH 39166 reversibly and dose-dependently decreases acetylcholine release in the rat striatum.

    Science.gov (United States)

    Acquas, E; Di Chiara, G

    1999-11-03

    The effect of local application by reverse dialysis of the dopamine D(1) receptor antagonist (-)-trans-6,7,7a,8,9, 13b-exahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]-nap hto-[2, 1b]-azepine hydrochloride (SCH 39166) on acetylcholine release was studied in awake, freely moving rats implanted with concentric microdialysis probes in the dorsal striatum. In these experiments, the reversible acetylcholine esterase inhibitor, neostigmine, was added to the perfusion solution at two different concentrations, 0.01 and 0.1 microM. SCH 39166 (1, 5 and 10 microM), in the presence of 0.01 microM neostigmine, reversibly decreased striatal acetylcholine release (1 microM SCH 39166 by 8+/-4%; 5 microM SCH 39166 by 24+/-5%; 10 microM SCH 39166 by 27+/-7%, from basal). Similarly, SCH 39166, applied in the presence of a higher neostigmine concentration (0.1 microM), decreased striatal acetylcholine release by 14+/-4% at 1 microM, by 28+/-8% at 5 microM and by 30+/-5% at 10 microM, in a dose-dependent and time-dependent manner. These results are consistent with the existence of a facilitatory tone of dopamine on striatal acetylcholine transmission mediated by dopamine D(1) receptors located on striatal cholinergic interneurons.

  6. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber).

    Science.gov (United States)

    Jørgensen, Kristine B; Krogh-Jensen, Karen; Pickering, Darryl S; Kanui, Titus I; Abelson, Klas S P

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1-5 of the naked mole-rat were compared to that of the house mouse (Mus musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies with [(3)H]-N-methylscopolamine. The BLAST test revealed 95 % protein sequence homology showing the naked mole-rat to have the genetic potential to express all five muscarinic acetylcholine receptor subtypes. A significant reduction in pain behavior was demonstrated after administration of 8.4 mg/kg in the formalin test. Administration of 50 mg/kg VU0152100 resulted in a non-significant tendency towards antinociception. The antinociceptive effects were reversed by the muscarinic acetylcholine receptor antagonist atropine. Binding studies indicated presence of muscarinic acetylcholine receptors with a radioligand affinity comparable to that reported in mice. In conclusion, muscarinic acetylcholine receptor subtypes are present in the naked mole-rat and contribute to antinociception in the naked mole-rat.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    DEFF Research Database (Denmark)

    Jensen, Majbrit Myrup; Mikkelsen, Jens D.; Arvaniti, Maria

    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...... 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 frontal...

  9. Lynx1 and Aβ1-42 bind competitively to multiple nicotinic acetylcholine receptor subtypes

    DEFF Research Database (Denmark)

    Thomsen, Morten S; Arvaniti, Maria; Jensen, Majbrit M

    2016-01-01

    Lynx1 regulates synaptic plasticity in the brain by regulating nicotinic acetylcholine receptors (nAChRs). It is not known to which extent Lynx1 can bind to endogenous nAChR subunits in the brain or how this interaction is affected by Alzheimer's disease pathology. We apply affinity purification....... Incubation with Ws-Lynx1 decreases nicotine-mediated extracellular signal-regulated kinase phosphorylation in PC12 cells and striatal neurons, indicating that binding of Ws-Lynx1 is sufficient to inhibit signaling downstream of nAChRs. The effect of nicotine in PC12 cells is independent of α7 or α4β2 n...

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

    International Nuclear Information System (INIS)

    Waechtler, K.

    1985-01-01

    The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using 3 H-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)

  11. Changes in acetylcholine release from the chick retina are not associated with myopia development

    International Nuclear Information System (INIS)

    Vessey, K.A.; Cotriall, C.L.; McBrien, N.A.

    2002-01-01

    Full text: The effectiveness of muscarinic receptor antagonists in inhibiting myopia progression in animal models and humans implicates cholinergic signalling in ocular growth regulation. Therefore to determine if changes in the release of acetylcholine from the retina are involved in myopia development, the efflux of acetylcholine from the in vitro retina of normal and myopic chick eyes was investigated. Chicks were monocularly deprived (MD) of pattern vision with translucent occluders for 2 or 7 days and refractive error of MD groups and age matched normals was monitored using retinoscopy (n=6 each group). 3 H-choline-Cl (1 Ci in 7μL) was injected into the vitreous of each eye under 2.5% halothane anaesthesia. After 1hr, the eyes were enucleated, under terminal anaesthesia (sodium pentobarbital, 120 mg/kg, im). Retinas were flat-mounted on acetate filter discs and superfused with oxygenated physiological saline solution (PSS) for 30min at 0.4mL/min. Five baseline fractions were collected (B1-B5), then three stimulated fractions were collected in the presence of PSS containing 50mM KCl (K1-K3) at 2min intervals. 3 H-acetylcholine ( 3 H-ACh) in each fraction was quantified by liquid scintillation counting. Significant amounts of myopia were induced in MD eyes after 2 (-5.1±0.8D) and 7 days (-18.8±2.4D) relative to control eyes (paired t-test p 3 H-ACh release was 146±15% above basal levels (K2/B1%) from retinas of normal animals. After 2 days MD, there was no significant difference between KCl-evoked release of 3 H-ACh from deprived eyes (147 39%) compared to control eyes (198±61%, paired t-test, p=0.27) or the eyes of normal animals (ANOVA, p>0.5). Similar results were obtained following 7 days MD. The results demonstrate that evoked acetylcholine release from the chick retina of myopic eyes is unaltered relative to control or normal eyes using an in vitro approach. Copyright (2002) Australian Neuroscience Society

  12. Temperature effect on proximal to distal gradient of quantal release of acetylcholine at frog endplate

    Czech Academy of Sciences Publication Activity Database

    Samigullin, D.; Bukharaeva, E.; Nikolsky, E.; Vyskočil, František

    2003-01-01

    Roč. 28, 3-4 (2003), s. 507-514 ISSN 0364-3190 R&D Projects: GA AV ČR IAA7011902; GA ČR GA305/02/1333; GA ČR GA202/02/1213 Grant - others:RFBR(RU) 02/04/48901 Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003 Keywords : neuromuscular junction * acetylcholine release * temperature Subject RIV: ED - Physiology Impact factor: 1.511, year: 2003

  13. Characterization of the positive and negative inotropic effects of acetylcholine in the human myocardium

    OpenAIRE

    Du, Xiaoyi; Schoemaker, Regien; Bos, Egbert; Saxena, Pramod Ranjan

    1995-01-01

    textabstractIn the human isolated myocardium, acetylcholine (10−9 to 10−3 M) elicited a biphasic inotropic effect (a decrease in the lower and an increase in the higher concentration range) in atrial and a positive inotropic effect in ventricular trabeculae. However, under conditions of raised contractility achieved by exposure to noradrenaline (10−5 M), only negative inotropic effects were observed in both atria and ventricles. Atropine (10−6 M), but not propranolol (10−6 M), antagonized bot...

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

    Energy Technology Data Exchange (ETDEWEB)

    Latli, B.; Casida, J.E. (California Univ., Berkeley, CA (United States). Dept. of Entomological Sciences)

    1992-08-01

    Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [[sup 3]H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB[sup 2]H[sub 4] (in model studies) or NaB[sup 3]H[sub 4] 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 [[sup 2]H[sub 2

  15. INFLUENCE OF ANTIBIOTICS ON THE MECHANICAL RESPONSES OF GUINEA-PIG ILEUM TO ACETYLCHOLINE AND HISTAMINE

    Directory of Open Access Journals (Sweden)

    Petroianu Andy

    1998-01-01

    Full Text Available The side effects of antibiotics have been extensively described during the last decades, however, their role on digestive motility must be better investigated. Following a line of research, the influence of penicillin, chloranfenicol tetracycline and gentamicine on longitudinal smooth muscle responses to acetylcholine and histamine were studied on guinea-pig ileum. There were no differences between the responses before and after the addition of each antibiotic. Further investigations must be performed in order to find a possible influence of antibiotics on digestive motility.

  16. Drugs interfering with Muscarinic Acetylcholine Receptors and Their effects on Place Navigation

    Czech Academy of Sciences Publication Activity Database

    Svoboda, Jan; Popelíková, Anna; Stuchlík, Aleš

    2017-01-01

    Roč. 8, Nov 9 (2017), č. článku 215. ISSN 1664-0640 R&D Projects: GA ČR(CZ) GA17-04047S; GA ČR NV17-30833A; GA MŠk(CZ) LM2015062 Grant - others:AV ČR(CZ) PAN-17-07 Program:Bilaterální spolupráce Institutional support: RVO:67985823 Keywords : scopolamine * biperiden * acetylcholine * receptor * behavior * learning Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 3.532, year: 2016

  17. Muscarinic Acetylcholine Receptor M3 Mutation Causes Urinary Bladder Disease and a Prune-Belly-like Syndrome.

    Science.gov (United States)

    Weber, Stefanie; Thiele, Holger; Mir, Sevgi; Toliat, Mohammad Reza; Sozeri, Betül; Reutter, Heiko; Draaken, Markus; Ludwig, Michael; Altmüller, Janine; Frommolt, Peter; Stuart, Helen M; Ranjzad, Parisa; Hanley, Neil A; Jennings, Rachel; Newman, William G; Wilcox, Duncan T; Thiel, Uwe; Schlingmann, Karl Peter; Beetz, Rolf; Hoyer, Peter F; Konrad, Martin; Schaefer, Franz; Nürnberg, Peter; Woolf, Adrian S

    2011-11-11

    Urinary bladder malformations associated with bladder outlet obstruction are a frequent cause of progressive renal failure in children. We here describe a muscarinic acetylcholine receptor M3 (CHRM3) (1q41-q44) homozygous frameshift mutation in familial congenital bladder malformation associated with a prune-belly-like syndrome, defining an isolated gene defect underlying this sometimes devastating disease. CHRM3 encodes the M3 muscarinic acetylcholine receptor, which we show is present in developing renal epithelia and bladder muscle. These observations may imply that M3 has a role beyond its known contribution to detrusor contractions. This Mendelian disease caused by a muscarinic acetylcholine receptor mutation strikingly phenocopies Chrm3 null mutant mice. Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  18. Autonomous control of phosphatidylinositol turnover by histamine and acetylcholine receptors in the NIE-115 neuron-like cell line

    International Nuclear Information System (INIS)

    Large, T.H.; Lambert, M.P.; Cohen, N.M.; Klein, W.L.

    1986-01-01

    Histamine was found to stimulate the turnover of phosphatidylinositol (PI) in cultures of neuron-like NE-115 cells. Turnover was measured by increased production of ( 3 H)inositol phosphates (breakdown) and by accelerated incorporation of 32 P into PI (resynthesis). Data were consistent with hydrolysis of polyphosphoinositides being the initial event in receptor-stimulated PI turnover. This response to histamine desensitized within 10 min. Receptor systems for histamine and acetylcholine were tested for possible interactions: PI turnover in response to dual stimulation was approximately equal to the sum of the individual responses while prior desensitization of the acetylcholine receptor system had no effect on subsequent stimulation of the histamine receptor system. These results are consistent with the hypothesis that components of acetylcholine and histamine receptor systems responsible for PI turnover are autonomously organised and regulated. (author)

  19. An extract of lionfish (Pterois volitans) spine tissue contains acetylcholine and a toxin that affects neuromuscular transmission.

    Science.gov (United States)

    Cohen, A S; Olek, A J

    1989-01-01

    A soluble toxic extract derived from spine tissue of the lionfish (Pterois volitans) decreased heart rate and force of contraction in isolated clam and frog hearts. These actions were due to the presence of micromolar concentrations of acetylcholine in the extract. Toxicity was retained after hydrolysis of acetylcholine by exogenous acetylcholinesterase, but heart function was no longer affected. Toxin treated in this way induced muscle fibrillation in an isolated nerve-muscle preparation, followed by blockade of neuromuscular transmission. Bursts of transient depolarizations were recorded at the muscle endplate shortly after toxin addition that correlated in time with the duration of toxin-induced muscle fibrillation. These effects are thought to be due to the increased release and then depletion of acetylcholine from the nerve terminal.

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

    DEFF Research Database (Denmark)

    Stromgaard, K; Brierley, M J; Andersen, K

    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...

  1. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    DEFF Research Database (Denmark)

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis

    2016-01-01

    of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-n...

  2. The A- and B-type muscarinic acetylcholine receptors from Drosophila melanogaster couple to different second messenger pathways

    DEFF Research Database (Denmark)

    Ren, Guilin Robin; Folke, Jonas; Hauser, Frank

    2015-01-01

    Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors (GPCRs) that are activated by the agonists acetylcholine and muscarine and blocked by several antagonists, among them atropine. In mammals five mAChRs (m1-m5) exist of which m1, m3, and m5 are coupled to members of the Gq...

  3. Effects of BMS-902483, an α7 nicotinic acetylcholine receptor partial agonist, on cognition and sensory gating in relation to receptor occupancy in rodents.

    Science.gov (United States)

    Pieschl, Rick L; Miller, Regina; Jones, Kelli M; Post-Munson, Debra J; Chen, Ping; Newberry, Kimberly; Benitex, Yulia; Molski, Thaddeus; Morgan, Daniel; McDonald, Ivar M; Macor, John E; Olson, Richard E; Asaka, Yukiko; Digavalli, Siva; Easton, Amy; Herrington, James; Westphal, Ryan S; Lodge, Nicholas J; Zaczek, Robert; Bristow, Linda J; Li, Yu-Wen

    2017-07-15

    The α7 nicotinic acetylcholine receptor is thought to play an important role in human cognition. Here we describe the in vivo effects of BMS-902483, a selective potent α7 nicotinic acetylcholine receptor partial agonist, in relationship to α7 nicotinic acetylcholine receptor occupancy. BMS-902483 has low nanomolar affinity for rat and human α7 nicotinic acetylcholine receptors and elicits currents in cells expressing human or rat α7 nicotinic acetylcholine receptors that are about 60% of the maximal acetylcholine response. BMS-902483 improved 24h novel object recognition memory in mice with a minimal effective dose (MED) of 0.1mg/kg and reversed MK-801-induced deficits in a rat attentional set-shifting model of executive function with an MED of 3mg/kg. Enhancement of novel object recognition was blocked by the silent α7 nicotinic acetylcholine receptor agonist, NS6740, demonstrating that activity of BMS-902483 was mediated by α7 nicotinic acetylcholine receptors. BMS-902483 also reversed ketamine-induced deficits in auditory gating in rats, and enhanced ex vivo hippocampal long-term potentiation examined 24h after dosing in mice. Results from an ex vivo brain homogenate binding assay showed that α7 receptor occupancy ranged from 64% (novel object recognition) to ~90% (set shift and gating) at the MED for behavioral and sensory processing effects of BMS-902483. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.

    2009-01-01

    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

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

    International Nuclear Information System (INIS)

    Dwyer, B.P.

    1991-01-01

    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 [ 3 H]-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

  6. Critical Evaluation of Acetylcholine Determination in Rat Brain Microdialysates using Ion-Pair Liquid Chromatography with Amperometric Detection

    Directory of Open Access Journals (Sweden)

    Yvette Michotte

    2008-08-01

    Full Text Available Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column (1 mm internal diameter, conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM (mean ± SD, n = 30. The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.

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

    DEFF Research Database (Denmark)

    Armishaw, Christopher J; Singh, Narender; Medina-Franco, Jose L

    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 neurop......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...

  8. Differential Regulation of Receptor Activation and Agonist Selectivity by Highly Conserved Tryptophans in the Nicotinic Acetylcholine Receptor Binding Site

    OpenAIRE

    Williams, Dustin K.; Stokes, Clare; Horenstein, Nicole A.; Papke, Roger L.

    2009-01-01

    We have shown previously that a highly conserved Tyr in the nicotinic acetylcholine receptor (nAChR) ligand-binding domain (LBD) (α7 Tyr188 or α4 Tyr195) differentially regulates the activity of acetylcholine (ACh) and the α7-selective agonist 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4OH-GTS-21) in α4β2 and α7 nAChR. In this study, we mutated two highly conserved LBD Trp residues in human α7 and α4β2 and expressed the receptors in Xenopus laevis oocytes. α7 Re...

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

    DEFF Research Database (Denmark)

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

    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....

  10. Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

    International Nuclear Information System (INIS)

    Forman, S.A.; Miller, K.W.

    1989-01-01

    The relationship between the high-affinity procaine channel inhibition site and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86 Rb + quenched-flux assays at 4 degree C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with α-bungarotoxin (α-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations alone, AChR undergoes one phase of inactivation in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting ACh concentrations rapid inactivation complete in 30-75 ms is followed by fast desensitization at the same k d observed without procaine. The dependence of k r on [procaine] is consistent with a bimolecular association between procaine and its AChR site. Inhibition of AChR function by mixtures of procaine plus self-inhibiting concentrations of ACh or suberyldicholine was studied by reducing the level of α-BTX block in vesicles. The data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and k r 's dependence on [ACh] in channel-activating range closely parallels that of 86 Rb + flux response to ACh

  11. cap alpha. -bungarotoxin binding properties of a central nervous system nicotinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lukasiewicz, R J; Bennett, E L

    1978-01-01

    High-affinity, specific binding of radiolabeled ..cap alpha..-bungarotoxin to particulate fractions derived from rat brain shows saturability (B/sub max/ approx. = 37fmol/mg, K/sub D//sup app/ = 1.7 nM) and insensitivity to ionic strength, and is essentially irreversible (K/sub on/ = 5 x 10/sup 6/ min/sup -1/ x mol/sup -1/; K(displacement) = 1.9 x 10/sup -4/ min/sup -1/, tau/sub 1/2/ = 62 h). Subcellular distribution of specific sites is consistent with their location on synaptic junctional complex and post-synaptic membranes. These membrane-bound binding sites exhibit unique sensitivity to cholinergic ligands; pretreatment of membranes with cholinergic agonists (but not antagonists) induces transformation of ..cap alpha..-bungarotoxin binding sites to a high affinity form toward agonist. The effect is most marked for the natural agonist, acetylcholine. These results strongly support the notion that the entity under study is an authentic nicotinic acetylcholine receptor.

  12. Acetylcholine-induced inhibition of presynaptic calcium signals and transmitter release in the frog neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Eduard Khaziev

    2016-12-01

    Full Text Available Acetylcholine (ACh, released from axonal terminals of motor neurones in neuromuscular junctions regulates the efficacy of neurotransmission through activation of presynaptic nicotinic and muscarinic autoreceptors. Receptor-mediated presynaptic regulation could reflect either direct action on exocytotic machinery or modulation of Ca2+ entry and resulting intra-terminal Ca2+ dynamics. We have measured free intra-terminal cytosolic Ca2+ ([Ca2+]i using Oregon-Green 488 microfluorimetry, in parallel with voltage-clamp recordings of spontaneous (mEPC and evoked (EPC postsynaptic currents in post-junctional skeletal muscle fibre. Activation of presynaptic muscarinic and nicotinic receptors with exogenous acetylcholine and its non-hydrolized analogue carbachol reduced amplitude of the intra-terminal [Ca2+]i transients and decreased quantal content (calculated by dividing the area under EPC curve by the area under mEPC curve. Pharmacological analysis revealed the role of muscarinic receptors of M2 subtype as well as d-tubocurarine-sensitive nicotinic receptor in presynaptic modulation of [Ca2+]i transients. Modulation of synaptic transmission efficacy by ACh receptors was completely eliminated by pharmacological inhibition of N-type Ca2+ channels. We conclude that ACh receptor-mediated reduction of Ca2+ entry into the nerve terminal through N-type Ca2+ channels represents one of possible mechanism of presynaptic modulation in frog neuromuscular junction.

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

    Directory of Open Access Journals (Sweden)

    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. Conformational changes in acetylcholine binding protein investigated by temperature accelerated molecular dynamics.

    Directory of Open Access Journals (Sweden)

    Zeynab Mohammad Hosseini Naveh

    Full Text Available Despite the large number of studies available on nicotinic acetylcholine receptors, a complete account of the mechanistic aspects of their gating transition in response to ligand binding still remains elusive. As a first step toward dissecting the transition mechanism by accelerated sampling techniques, we study the ligand-induced conformational changes of the acetylcholine binding protein (AChBP, a widely accepted model for the full receptor extracellular domain. Using unbiased Molecular Dynamics (MD and Temperature Accelerated Molecular Dynamics (TAMD simulations we investigate the AChBP transition between the apo and the agonist-bound state. In long standard MD simulations, both conformations of the native protein are stable, while the agonist-bound structure evolves toward the apo one if the orientation of few key sidechains in the orthosteric cavity is modified. Conversely, TAMD simulations initiated from the native conformations are able to produce the spontaneous transition. With respect to the modified conformations, TAMD accelerates the transition by at least a factor 10. The analysis of some specific residue-residue interactions points out that the transition mechanism is based on the disruption/formation of few key hydrogen bonds. Finally, while early events of ligand dissociation are observed already in standard MD, TAMD accelerates the ligand detachment and, at the highest TAMD effective temperature, it is able to produce a complete dissociation path in one AChBP subunit.

  15. Molecular determinants of subtype-selective efficacies of cytisine and the novel compound NS3861 at heteromeric nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Harpsøe, Kasper; Hald, Helle; Timmermann, Daniel B

    2013-01-01

    Deciphering which specific agonist-receptor interactions affect efficacy levels is of high importance, because this will ultimately aid in designing selective drugs. The novel compound NS3861 and cytisine are agonists of nicotinic acetylcholine receptors (nAChRs) and both bind with high affinity...

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    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...

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

    DEFF Research Database (Denmark)

    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...

  18. Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

    Directory of Open Access Journals (Sweden)

    Monica S Guzman

    2011-11-01

    Full Text Available Cholinergic neurons in the striatum are thought to play major regulatory functions in motor behaviour and reward. These neurons express two vesicular transporters that can load either acetylcholine or glutamate into synaptic vesicles. Consequently cholinergic neurons can release both neurotransmitters, making it difficult to discern their individual contributions for the regulation of striatal functions. Here we have dissected the specific roles of acetylcholine release for striatal-dependent behaviour in mice by selective elimination of the vesicular acetylcholine transporter (VAChT from striatal cholinergic neurons. Analysis of several behavioural parameters indicates that elimination of VAChT had only marginal consequences in striatum-related tasks and did not affect spontaneous locomotion, cocaine-induced hyperactivity, or its reward properties. However, dopaminergic sensitivity of medium spiny neurons (MSN and the behavioural outputs in response to direct dopaminergic agonists were enhanced, likely due to increased expression/function of dopamine receptors in the striatum. These observations indicate that previous functions attributed to striatal cholinergic neurons in spontaneous locomotor activity and in the rewarding responses to cocaine are mediated by glutamate and not by acetylcholine release. Our experiments demonstrate how one population of neurons can use two distinct neurotransmitters to differentially regulate a given circuitry. The data also raise the possibility of using VAChT as a target to boost dopaminergic function and decrease high striatal cholinergic activity, common neurochemical alterations in individuals affected with Parkinson's disease.

  19. The Actions of Piperidine Alkaloids at Fetal Muscle-Type and Autonomic-Type Nicotinic Acetylcholine Receptors

    Science.gov (United States)

    Piperidine alkaloids are found in many species of plants including Conium maculatum, Nicotiana spp., and Lupinus spp. A pharmacodynamic comparison was made of the alkaloids ammodendrine, anabasine, anabaseine, and coniine in; SH-SY5Y cells which express autonomic-type nicotinic acetylcholine recept...

  20. NMR Structure and Action on Nicotinic Acetylcholine Receptors of Water-soluble Domain of Human LYNX1

    Czech Academy of Sciences Publication Activity Database

    Lyukmanova, E. N.; Shenkarev, Z. O.; Shulepko, M. A.; Mineev, K. S.; D´Hoedt, D.; Kasheverov, I. E.; Filkin, S. Yu.; Krivolapova, A. P.; Janíčková, Helena; Doležal, Vladimír; Dolgikh, D. A.; Arseniev, A. S.; Bertrand, D.; Tsetlin, V.I.; Kirpichnikov, M. P.

    2011-01-01

    Roč. 286, č. 12 (2011), s. 10618-10627 ISSN 0021-9258 R&D Projects: GA ČR(CZ) GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : NMR structure * nicotinic acetylcholine receptor * water-soluble domain Subject RIV: FH - Neurology Impact factor: 4.773, year: 2011

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

    NARCIS (Netherlands)

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

    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

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

    DEFF Research Database (Denmark)

    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...

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

    Czech Academy of Sciences Publication Activity Database

    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

  4. Intravenously administered oxotremorine and atropine, in doses known to affect pain threshold, affect the intraspinal release of acetylcholine in rats

    DEFF Research Database (Denmark)

    Abelson, Klas S P; Höglund, A Urban

    2002-01-01

    muscarinic agonists and antagonists modify nociceptive threshold by affecting intraspinal release of acetylcholine (ACh). Catheters were inserted into the femoral vein in rats maintained on isoflurane anaesthesia for administration of oxotremorine (10-300 microg/kg) and atropine (0.1, 10, 5000 microg...

  5. Activation and desensitization of peripheral muscle and neuronal nicotinic acetylcholine receptors by selected, naturally-occurring pyridine alkaloids

    Science.gov (United States)

    Teratogenic alkaloids can cause developmental defects due to inhibition of fetal movement that results from desensitization of fetal muscletype nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiper...

  6. Allosteric modulation by persistent binding of xanomeline of the interaction of competitive ligands with the M1 muscarinic acetylcholine receptor

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; Tuček, Stanislav; El-Fakahany, E. E.

    2002-01-01

    Roč. 301, č. 3 (2002), s. 1033-1041 ISSN 0022-3565 R&D Projects: GA ČR GP305/01/D119 Grant - others:NIH(US) NS25743 Institutional research plan: CEZ:AV0Z5011922 Keywords : xanomeline * M(1) muscarinic acetylcholine receptor Subject RIV: FH - Neurology Impact factor: 3.991, year: 2002

  7. Cholinesterase inhibition and acetylcholine accumulation following intracerebral administration of paraoxon in rats

    International Nuclear Information System (INIS)

    Ray, A.; Liu, J.; Karanth, S.; Gao, Y.; Brimijoin, S.; Pope, C.

    2009-01-01

    We evaluated the inhibition of striatal cholinesterase activity following intracerebral administration of paraoxon assaying activity either in tissue homogenates ex vivo or by substrate hydrolysis in situ. Artificial cerebrospinal fluid (aCSF) or paraoxon in aCSF was infused unilaterally (0.5 μl/min for 2 h) and ipsilateral and contralateral striata were harvested for ChE assay ex vivo. High paraoxon concentrations were needed to inhibit ipsilateral striatal cholinesterase activity (no inhibition at < 0.1 mM; 27% at 0.1 mM; 79% at 1 mM paraoxon). With 3 mM paraoxon infusion, substantial ChE inhibition was also noted in contralateral striatum. ChE histochemistry generally confirmed these concentration- and side-dependent effects. Microdialysates collected for up to 4 h after paraoxon infusion inhibited ChE activity when added to striatal homogenate, suggesting prolonged efflux of paraoxon. Since paraoxon efflux could complicate acetylcholine analysis, we evaluated the effects of paraoxon (0, 0.03, 0.1, 1, 10 or 100 μM, 1.5 μl/min for 45 min) administered by reverse dialysis through a microdialysis probe. ChE activity was then monitored in situ by perfusing the colorimetric substrate acetylthiocholine through the same probe and measuring product (thiocholine) in dialysates. Concentration-dependent inhibition was noted but reached a plateau of about 70% at 1 μM and higher concentrations. Striatal acetylcholine was below the detection limit at all times with 0.1 μM paraoxon but was transiently elevated (0.5-1.5 h) with 10 μM paraoxon. In vivo paraoxon (0.4 mg/kg, sc) in adult rats elicited about 90% striatal ChE inhibition measured ex vivo, but only about 10% inhibition measured in situ. Histochemical analyses revealed intense AChE and glial fibrillary acidic protein staining near the cannula track, suggesting proliferation of inflammatory cells/glia. The findings suggest that ex vivo and in situ cholinesterase assays can provide very different views into enzyme

  8. Acetylcholine-induced vasodilation in the uterine vascular bed of pregnant rats with adriamycin-induced nephrosis.

    Science.gov (United States)

    Yousif, Mariam H; Adeagbo, Ayotunde S; Kadavil, Elizabeth A; Chandrasekhar, Bindu; Oriowo, Mabayoje A

    2002-01-01

    This project was designed to study endothelium-dependent vasodilation in the uterine vascular bed during experimentally induced preeclampsia in rats. Uterine vascular beds were isolated from non-pregnant and pregnant rats with or without treatment with adriamycin (ADR) and perfused with physiological solution. Thereafter, vasodilator responses to acetylcholine were recorded. RECORDS: Pregnant ADR-treated rats displayed symptoms of preeclampsia including hypertension and proteinuria. Blood pressure was 110.0 +/- 4.7 mm Hg (n = 5) in control pregnant rats and 136.0 +/- 5.3 mm Hg (n = 5) in ADR-treated pregnant rats, and urinary protein concentrations were 0.35 mg/ml (n = 5) and 13.2 +/- 3.6 mg/ml (n = 9), respectively. Both blood pressure and proteinuria values were significantly (p acetylcholine-induced dose-dependent vasodilator responses in the vascular beds were not significantly different between the pregnant and nonpregnant rats. Although acetylcholine-induced vasodilation was significantly reduced by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) in both groups, the residual response to acetylcholine was not affected by indomethacin, suggesting that prostanoids were not involved in this response. The L-NAME-resistant component, endothelium-derived hyperpolarizing factor (EDHF), was greater in ADR-treated uterine beds than in those of the controls, indicating a significant contribution from EDHF in these vessels. In the presence of an elevated external potassium ion concentration, acetylcholine produced similar vasodilator responses, indicating that the release of nitric oxide was not impaired. These results indicate that endothelium-dependent vasodilation was not impaired in this model of preeclampsia.

  9. Expression of nicotinic acetylcholine receptors on human B-lymphoma cells

    Directory of Open Access Journals (Sweden)

    Skok M. V.

    2009-12-01

    Full Text Available Aim. To find a correlation between the level of nicotinic acetylcholine receptor (nAChR expression and B lymphocyte differentiation or activation state. Methods. Expression of nAChRs in the REH, Ramos and Daudi cell lines was studied by flow cytometry using nAChR subunit-specific antibodies; cell proliferation was studied by MTT test. Results. It is shown that the level of 42/4 and 7 nAChRs expression increased along with B lymphocyte differentiation (Ramos > REH and activation (Daudi > > Ramos and depended on the antigen-specific receptor expression. The nAChR stimulation/blockade did not influence the intensity of cell proliferation.

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

    International Nuclear Information System (INIS)

    Kaplan, I.; Blakely, B.T.; Pavlath, G.K.; Travis, M.; Blau, H.M.

    1990-01-01

    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

  11. Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

    Science.gov (United States)

    Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

    2011-08-15

    Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. The Nicotinic Acetylcholine Receptor as a Target for Antidepressant Drug Development

    Directory of Open Access Journals (Sweden)

    Noah S. Philip

    2012-01-01

    Full Text Available An important new area of antidepressant drug development involves targeting the nicotinic acetylcholine receptor (nAChR. This receptor, which is distributed widely in regions of the brain associated with depression, is also implicated in other important processes that are relevant to depression, such as stress and inflammation. The two classes of drugs that target nAChRs can be broadly divided into mecamylamine- and cytisine-based compounds. These drugs probably exert their effects via antagonism at α4β2 nAChRs, and strong preclinical data support the antidepressant efficacy of both classes when used in conjunction with other primary antidepressants (e.g., monoamine reuptake inhibitors. Although clinical data remain limited, preliminary results in this area constitute a compelling argument for further evaluation of the nAChR as a target for future antidepressant drug development.

  13. Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain

    DEFF Research Database (Denmark)

    Wu, Jie; Liu, Qiang; Tang, Pei

    2016-01-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule...... governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co...... nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling....

  14. Molecular basis of the functional heterogeneity of the muscarinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Numa, S.; Fukuda, K.; Kubo, T.; Maeda, A.; Akiba, I.; Bujo, H.; Nakai, J.; Mishina, M.; Higashida, H.

    1988-01-01

    The muscarinic acetylcholine receptor (mAChR) mediates a variety of cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides, and modulation of potassium channels, through the action of guanine-nucleotide-binding regulatory proteins (G proteins). The question then arises as to whether multiple mAChR species exist that are responsible for the various biochemical and physiological effects. In fact, pharmacologically distinguishable forms of the mAChR occur in different tissues and have been provisionally classified into M 1 (I), M 2 cardiac (II), and M 2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. Here, the authors have made attempts to understand the molecular basis of the functional heterogeneity of the mAChR, using recombinant DNA technology

  15. [3H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Latli, B.; Casida, J.E.

    1992-01-01

    Imidacloprid is an exceptionally potent insecticide known from physiological studies to act at the nicotinic acetylcholine receptor. To prepare [ 3 H]imidacloprid as a candidate radioligand, 6-chloronicotinoyl chloride was reduced with NaB 2 H 4 (in model studies) or NaB 3 H 4 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 [ 2 H 2 ]imidacloprid incorporating about 95% of the deuterium or [ 3 H 2 ]imidacloprid (25 Ci/mmol) in 80% radiochemical yield. In studies not detailed here [ 3 H] imidacloprid was found to undergo high affinity, specific and saturable binding to a site in insect brain. (author)

  16. Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

    Science.gov (United States)

    Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

    2004-01-01

    In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

  17. Reactivity of Acetylcholine Esterase in inner Ear Maculae of Fish after Development at Hypergravity

    Science.gov (United States)

    Feucht, I.; Hilbig, R.; Anken, R.

    It has been shown earlier that the growth of inner ear otoliths of larval fish is (among other environmental factors) guided by the gravity vector. This guidance most probably is effected by the efferent vestibular system in the brainstem, because a transection of the nervus vestibularis has been shown to effect a cessation of the supply of calcium to the otoliths. The efferent innervation of fish inner ear maculae uses the synaptic transmitter acetylcholine (ACh). Therefore, we were - in order to further assess the role of the efferent system for otolith growth - prompted to determine ACh esterase-reactivity in the sensory epithelium of the utricle and the saccule (as well as in a non-gravity relevant brain region for control) in larval cichlid fish (Oreochromis mossambicus), which had been maintained at hypergravity during their development. The respective data will be communicated at the meeting. Acknowledgement: This work was financially supported by the German Aerospace Center (DLR) (FKZ: 50 WB 9997).

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Acetylcholine causes rooting in leaf explants of in vitro raised tomato (Lycopersicon esculentum Miller) seedlings.

    Science.gov (United States)

    Bamel, Kiran; Gupta, Shrish Chandra; Gupta, Rajendra

    2007-05-30

    The animal neurotransmitter acetylcholine (ACh) induces rooting and promotes secondary root formation in leaf explants of tomato (Lycopersicon esculentum Miller var. Pusa Ruby), cultured in vitro on Murashige and Skoog's medium. The roots originate from the midrib of leaf explants and resemble taproot. ACh at 10(-5) M was found to be the optimum over a wide range of effective concentrations between 10(-7) and 10(-3) M. The breakdown products, choline and acetate were ineffective even at 10(-3) M concentration. ACh appears to have a natural role in tomato rhizogenesis because exogenous application of neostigmine, an inhibitor of ACh hydrolysis, could mimic the effect of ACh. Neostigmine, if applied in combination with ACh, potentiated the ACh effect.

  20. Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs.

    Directory of Open Access Journals (Sweden)

    Nikita Abraham

    Full Text Available Nicotinic acetylcholine receptors (nAChR are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP. AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies.

  1. Characterization of prejunctional serotonin receptors modulating [3H]acetylcholine release in the human detrusor.

    Science.gov (United States)

    D'Agostino, Gianluigi; Condino, Anna M; Gallinari, Paola; Franceschetti, Gian P; Tonini, Marcello

    2006-01-01

    Bladder overactivity (OAB) is a chronic and debilitating lower urinary tract (LUT) disorder that affects millions of individuals worldwide. LUT symptoms associated with OAB, such as urgency and urinary incontinence, cause a hygienic and social concern to patients, but their current pharmacological treatment is largely inadequate due to the lack of uroselectivity. Although OAB etiology remains multifactorial and poorly understood, increasing evidence indicates that serotonin [5-hydroxytryptamine (5-HT)] is an endogenous substance involved in the control of micturition at central and peripheral sites. In this study, we demonstrated the presence of three distinct 5-HT receptors localized at parasympathetic nerve terminals of the human bladder by measuring electrically evoked tritiated acetylcholine release in isolated detrusor strips. These prejunctional receptors, involved in both positive and negative feedback mechanisms regulating cholinergic transmission, have been characterized by means of three highly selective 5-HT antagonists for 5-HT(4), 5-HT(7), and 5-HT(1A) receptors, namely GR113808A ([1-[2-[(-methylsulphonyl) amino] ethyl]4-piperinidyl]methyl1-methyl-1H-indole-3-carboxylate succinate), SB269970 [(R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)pyrrolidine-1-sulfonyl)phenol hydrochloride], and WAY100635 [N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridyl)-cyclohexane-carboxamide trichloride]. Under these conditions, we confirmed the facilitatory role of 5-HT(4) heteroreceptors on acetylcholine release and revealed for the first time the occurrence of 5-HT(7) and 5-HT(1A) heteroreceptors with a facilitatory and an inhibitory action, respectively. Our findings strengthen the novel concept for the use of recently patented selective 5-HT agonists and antagonists for the control of OAB dysfunctions associated with inflammatory conditions, although their therapeutic efficacy needs to be explored in the clinical setting.

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

    International Nuclear Information System (INIS)

    Brown, Derek; Chisholm, Jennifer A.; Patterson, Jim; Wyper, David; Owens, Jonathan; Pimlott, Sally

    2003-01-01

    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)[ 123 I]I-quinuclidinyl benzilate (R,R[ 123 I]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)[ 123 I]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)[ 123 I]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)[ 123 I]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.)

  3. Carbon monoxide-induced delayed amnesia, delayed neuronal death and change in acetylcholine concentration in mice

    International Nuclear Information System (INIS)

    Nabeshima, T.; Katoh, A.; Ishimaru, H.; Yoneda, Y.; Ogita, K.; Murase, K.; Ohtsuka, H.; Inari, K.; Fukuta, T.; Kameyama, T.

    1991-01-01

    We investigated the interrelationship of delayed amnesia, delayed neuronal death and changes in acetylcholine concentration induced by carbon monoxide (CO)-exposure in mice. In the test for retention of the passive avoidance task, amnesia was observed 5 and 7 days after CO-exposure when the mice were exposed to CO 1 day after training; in the case when the mice were exposed to CO 5 and 7 days before training, amnesia was also observed in a retention test given 1 day after training. The number of pyramidal cells in the hippocampal CA1 subfield was lower than that of the control 3, 5 and 7 days after CO-exposure. But the neurodegeneration in the parietal cortex, area 1, was not observed until 7 days after CO-exposure. The findings indicated that the amnesia and the neuronal death were produced after a delay when the mice were exposed to CO. In addition, the delayed amnesia was closely related to the delayed neuronal death in the hippocampal CA1 subfield. Moreover, [3H]glutamate and [3H]glycine binding sites did not change after CO-exposure but, 7 days after CO-exposure, the concentration of acetylcholine and the binding of [3H]quinuclidinyl benzilate in the frontal cortex and the striatum were found to have significantly changed, but those in the hippocampus did not show significant change. Therefore, we suggest that delayed amnesia induced by CO-exposure may result from delayed neuronal death in the hippocampal CA1 subfield and dysfunction in the acetylcholinergic neurons, in the frontal cortex, the striatum and/or the hippocampus

  4. Carbon monoxide-induced delayed amnesia, delayed neuronal death and change in acetylcholine concentration in mice

    Energy Technology Data Exchange (ETDEWEB)

    Nabeshima, T.; Katoh, A.; Ishimaru, H.; Yoneda, Y.; Ogita, K.; Murase, K.; Ohtsuka, H.; Inari, K.; Fukuta, T.; Kameyama, T. (Meijo Univ., Nagoya (Japan))

    1991-01-01

    We investigated the interrelationship of delayed amnesia, delayed neuronal death and changes in acetylcholine concentration induced by carbon monoxide (CO)-exposure in mice. In the test for retention of the passive avoidance task, amnesia was observed 5 and 7 days after CO-exposure when the mice were exposed to CO 1 day after training; in the case when the mice were exposed to CO 5 and 7 days before training, amnesia was also observed in a retention test given 1 day after training. The number of pyramidal cells in the hippocampal CA1 subfield was lower than that of the control 3, 5 and 7 days after CO-exposure. But the neurodegeneration in the parietal cortex, area 1, was not observed until 7 days after CO-exposure. The findings indicated that the amnesia and the neuronal death were produced after a delay when the mice were exposed to CO. In addition, the delayed amnesia was closely related to the delayed neuronal death in the hippocampal CA1 subfield. Moreover, (3H)glutamate and (3H)glycine binding sites did not change after CO-exposure but, 7 days after CO-exposure, the concentration of acetylcholine and the binding of (3H)quinuclidinyl benzilate in the frontal cortex and the striatum were found to have significantly changed, but those in the hippocampus did not show significant change. Therefore, we suggest that delayed amnesia induced by CO-exposure may result from delayed neuronal death in the hippocampal CA1 subfield and dysfunction in the acetylcholinergic neurons, in the frontal cortex, the striatum and/or the hippocampus.

  5. Effect of ethanol at clinically relevant concentrations on atrial inward rectifier potassium current sensitive to acetylcholine.

    Science.gov (United States)

    Bébarová, Markéta; Matejovič, Peter; Pásek, Michal; Hořáková, Zuzana; Hošek, Jan; Šimurdová, Milena; Šimurda, Jiří

    2016-10-01

    Alcohol intoxication tends to induce arrhythmias, most often the atrial fibrillation. To elucidate arrhythmogenic mechanisms related to alcohol consumption, the effect of ethanol on main components of the ionic membrane current is investigated step by step. Considering limited knowledge, we aimed to examine the effect of clinically relevant concentrations of ethanol (0.8-80 mM) on acetylcholine-sensitive inward rectifier potassium current I K(Ach). Experiments were performed by the whole-cell patch clamp technique at 23 ± 1 °C on isolated rat and guinea-pig atrial myocytes, and on expressed human Kir3.1/3.4 channels. Ethanol induced changes of I K(Ach) in the whole range of concentrations applied; the effect was not voltage dependent. The constitutively active component of I K(Ach) was significantly increased by ethanol with the maximum effect (an increase by ∼100 %) between 8 and 20 mM. The changes were comparable in rat and guinea-pig atrial myocytes and also in expressed human Kir3.1/3.4 channels (i.e., structural correlate of I K(Ach)). In the case of the acetylcholine-induced component of I K(Ach), a dual ethanol effect was apparent with a striking heterogeneity of changes in individual cells. The effect correlated with the current magnitude in control: the current was increased by eth-anol in the cells showing small current in control and vice versa. The average effect peaked at 20 mM ethanol (an increase of the current by ∼20 %). Observed changes of action potential duration agreed well with the voltage clamp data. Ethanol significantly affected both components of I K(Ach) even in concentrations corresponding to light alcohol consumption.

  6. Adenosine receptors and muscarinic receptors cooperate in acetylcholine release modulation in the neuromuscular synapse.

    Science.gov (United States)

    Santafe, M M; Priego, M; Obis, T; Garcia, N; Tomàs, M; Lanuza, M A; Tomàs, J

    2015-07-01

    Adenosine receptors (ARs) are present in the motor terminals at the mouse neuromuscular junction. ARs and the presynaptic muscarinic acetylcholine receptors (mAChRs) share the functional control of the neuromuscular junction. We analysed their mutual interaction in transmitter release modulation. In electrophysiological experiments with unaltered synaptic transmission (muscles paralysed by blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB), we found that: (i) a collaborative action between different AR subtypes reduced synaptic depression at a moderate activity level (40 Hz); (ii) at high activity levels (100 Hz), endogenous adenosine production in the synaptic cleft was sufficient to reduce depression through A1 -type receptors (A1 Rs) and A2 A-type receptors (A2 A Rs); (iii) when the non-metabolizable 2-chloroadenosine (CADO) agonist was used, both the quantal content and depression were reduced; (iv) the protective effect of CADO on depression was mediated by A1 Rs, whereas A2 A Rs seemed to modulate A1 Rs; (v) ARs and mAChRs absolutely depended upon each other for the modulation of evoked and spontaneous acetylcholine release in basal conditions and in experimental conditions with CADO stimulation; (vi) the purinergic and muscarinic mechanisms cooperated in the control of depression by sharing a common pathway although the purinergic control was more powerful than the muscarinic control; and (vii) the imbalance of the ARs created by using subtype-selective and non-selective inhibitory and stimulatory agents uncoupled protein kinase C from evoked transmitter release. In summary, ARs (A1 Rs, A2 A Rs) and mAChRs (M1 , M2 ) cooperated in the control of activity-dependent synaptic depression and may share a common protein kinase C pathway. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

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

    Science.gov (United States)

    González, Alfredo; Crittenden, Elizabeth L; García, Dana M

    2004-07-13

    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. 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. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Linzy M. Hendrickson

    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.

  10. Modulation of release of [3H]acetylcholine in the major pelvic ganglion of the rat.

    Science.gov (United States)

    Somogyi, G T; de Groat, W C

    1993-06-01

    Cholinergic modulation of [3H]acetylcholine release evoked by electrical stimulation was studied in the rat major pelvic ganglion, which was prelabeled with [3H]choline. Acetylcholine (ACh) release was independent of the frequency of stimulation; 0.3 Hz produced the same volley output as 10 Hz. Tetrodotoxin (1 microM) or omission of Ca2+ from the medium abolished ACh release. The M1 receptor agonist (4-hydroxy-2-butynyl)-1-trimethylammonium m-chlorocarbanilate chloride (McN-A 343, 50 microM) increased release (by 136%), whereas the M2 muscarinic agonist oxotremorine (1 microM) decreased ACh release (by 22%). The muscarinic antagonists, atropine (1 microM) or pirenzepine (M1 selective, 1 microM), did not change ACh release. However, pirenzepine (1 microM) blocked the facilitatory effect of McN-A 343, and atropine (1 microM) blocked the inhibitory effect of oxotremorine. The cholinesterase inhibitor physostigmine (1-5 microM), the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP, 10 microM), and the nicotinic antagonist D-tubocurarine (50 microM) did not change ACh release. 4-Aminopyridine, a K+ channel blocker, significantly increased the release (by 146%). Seven days after decentralization of the major pelvic ganglion, the evoked release of ACh was abolished. It is concluded that release of ACh occurs from the preganglionic nerve terminals rather than from the cholinergic cell bodies and is not modulated by actions of endogenous ACh on either muscarinic or nicotinic autoreceptors. These data confirm and extend previous electrophysiological findings indicating that synapses in the major pelvic ganglion have primarily a relay function.

  11. Modes of Action, Resistance and Toxicity of Insecticides Targeting Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Ihara, Makoto; Buckingham, Steven D; Matsuda, Kazuhiko; Sattelle, David B

    2017-01-01

    Nicotinic acetylcholine receptors (nAChRs) of insects play a key role in fast excitatory neurotransmission. Several classes of insecticides target insect nAChRs, which are composed of subunit members of a family of multiple subunit encoding genes. Alternative splicing and RNA A-to-I editing can add further to receptor diversity. Native and recombinant receptors have been explored as sites of insecticide action using radioligands, electrophysiology and site-directed mutagenesis. We have reviewed the properties of native and recombinant insect nAChRs, the challenges of functional recombinant insect nAChR expression, nAChR interactions with ligands acting at orthosteric and allosteric sites and in particular their interactions with insecticides. Actions on insect nAChRs of cartap, neonicotinoids, spinosyns, sulfoxamines, butenolides and mesoionic insecticides are reviewed and current knowledge of their modes of action are addressed. Mutations that add to our understanding of insecticide action and those leading to resistance are discussed. Co-crystallisation of neonicotinoids with the acetylcholine binding protein (AChBP), a surrogate for the nAChR ligand binding domain, has proved instructive. Toxicity issues relating to insecticides targeting nAChRs are also considered. An overview of insecticide classes targeting insect nAChRs has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native nAChRs remains poorly understood and functional expression still presents difficulties. These topics together with improved understanding of the precise sites of insecticide actions on insect nAChRs will be the subject of future research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Super agonist actions of clothianidin and related compounds on the SAD beta 2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Ihara, Makoto; Matsuda, Kazuhiko; Shimomura, Masaru; Sattelle, David B; Komai, Koichiro

    2004-03-01

    To compare the actions of clothianidin, a neonicotinoid acting on insect nicotinic acetylcholine receptors, and related compounds with that of imidacloprid, the compounds were tested on the Drosophila SAD-chicken beta2 nicotinic acetylcholine receptor expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. The maximum response of the SAD beta 2 nicotinic receptor to clothianidin was larger than that observed for acetylcholine. Ring breakage of the imidazolidine ring of imidacloprid resulting in the generation of a guanidine group was critical for this super agonist action.

  13. Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors.

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    Iwamoto, K; Mata, D; Linn, D M; Linn, C L

    2013-05-01

    Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double

  14. Ion Transport in Human Pancreatic Duct Epithelium, Capan-1 Cells, Is Regulated by Secretin, VIP, Acetylcholine, and Purinergic Receptors

    DEFF Research Database (Denmark)

    Wang, Jing; Novak, Ivana

    2013-01-01

    OBJECTIVES: The objective of the study was to establish a solid model of polarized epithelium for human pancreatic ducts, where electrical parameters could be measured as indicators of ion transport. Further, we aimed to determine functional expression of several receptors, in particular, puriner...... transport in human pancreatic duct epithelium, Capan-1 cells, is regulated by secretin, VIP, acetylcholine, adenosine, and purinergic P2 receptors; and this human model has a good potential for studies of physiology and pathophysiology of pancreatic duct ion transport....

  15. Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine

    OpenAIRE

    Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

    2015-01-01

    Background Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. Results We demonstrate the effectiveness of the small bioactive molecule ?acetylcholine? on accumulation of biomass, total li...

  16. Lipophilicity of acetylcholine and related ions examined by ion transfer voltammetry at a polarized room-temperature ionic liquid membrane

    Czech Academy of Sciences Publication Activity Database

    Langmaier, Jan; Záliš, Stanislav; Samec, Zdeněk

    2018-01-01

    Roč. 815, APR 2018 (2018), s. 183-188 ISSN 1572-6657 R&D Projects: GA ČR(CZ) GA15-03139S; GA ČR(CZ) GA17-05387S Institutional support: RVO:61388955 Keywords : acetylcholine * choline * ion transfer voltammetry Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 3.012, year: 2016

  17. Whole-Retina Reduced Electrophysiological Activity in Mice Bearing Retina-Specific Deletion of Vesicular Acetylcholine Transporter.

    Directory of Open Access Journals (Sweden)

    Jake Bedore

    Full Text Available Despite rigorous characterization of the role of acetylcholine in retinal development, long-term effects of its absence as a neurotransmitter are unknown. One of the unanswered questions is how acetylcholine contributes to the functional capacity of mature retinal circuits. The current study investigates the effects of disrupting cholinergic signalling in mice, through deletion of vesicular acetylcholine transporter (VAChT in the developing retina, pigmented epithelium, optic nerve and optic stalk, on electrophysiology and structure of the mature retina.A combination of electroretinography, optical coherence tomography imaging and histological evaluation assessed retinal integrity in mice bearing retina- targeted (embryonic day 12.5 deletion of VAChT (VAChTSix3-Cre-flox/flox and littermate controls at 5 and 12 months of age. VAChTSix3-Cre-flox/flox mice did not show any gross changes in nuclear layer cellularity or synaptic layer thickness. However, VAChTSix3-Cre-flox/flox mice showed reduced electrophysiological response of the retina to light stimulus under scotopic conditions at 5 and 12 months of age, including reduced a-wave, b-wave, and oscillatory potential (OP amplitudes and decreased OP peak power and total energy. Reduced a-wave amplitude was proportional to the reduction in b-wave amplitude and not associated with altered a-wave 10%-90% rise time or inner and outer segment thicknesses.This study used a novel genetic model in the first examination of function and structure of the mature mouse retina with disruption of cholinergic signalling. Reduced amplitude across the electroretinogram wave form does not suggest dysfunction in specific retinal cell types and could reflect underlying changes in the retinal and/or extraretinal microenvironment. Our findings suggest that release of acetylcholine by VAChT is essential for the normal electrophysiological response of the mature mouse retina.

  18. R+-methanandamide inhibits tracheal response to endogenously released acetylcholine via capsazepine-sensitive receptors.

    Science.gov (United States)

    Nieri, Paola; Martinotti, Enrica; Testai, Lara; Adinolfi, Barbara; Calderone, Vincenzo; Breschi, Maria Cristina

    2003-01-10

    The effects of cannabinoid drugs on the cholinergic response evoked by electrical field stimulation (0.2 ms pulse width, 20 V amplitude, 10 Hz, 7.5 s train duration) in guinea-pig tracheal preparations were investigated. The stable analogue of the endocannabinoid anandamide, R(+)-methanandamide (10(-7)-10(-4) M), produced a dose-dependent inhibition (up to 27+/-5% of control) of electrical field stimulation-mediated atropine-sensitive response. This effect was not blocked by the selective cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3 carboxamide hydrochloride (SR 141716A; 10(-6) M), and was not reproduced with the cannabinoid CB(1)/CB(2) receptor agonist R(+)-[2,3-dihydro-5-methyl-[(morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazin-6-yl]-(1-naphthalenyl)methanone mesylate) (WIN 55,212-2; 10(-8)-10(-5) M) or the cannabinoid CB(2) receptor selective agonist 1-propyl-2-methyl-3-(1-naphthoyl)indole (JWH-015; 10(-8)-10(-5) M); it was, on the contrary, antagonized by the vanilloid antagonist 2-[2-(4-chlorophenyl)ethyl-amino-thiocarbonyl]-7,8-dihydroxy-2,3,4,5-tetrahydro-1H-2 benzazepine (capsazepine; 10(-6) M). At the postjunctional level, neither R(+)-methanandamide nor WIN 55,212-2 nor JWH-015 did affect tracheal contractions induced by exogenous acetylcholine (10(-6) M). An inhibitory vanilloid receptor-mediated effect on the cholinergic response evoked by electrical stimulation was confirmed with the vanilloid agonist capsaicin, at doses (3-6 x 10(-8) M) which poorly influenced the basal smooth muscle tone of trachea. In conclusion, our data indicate that in guinea-pig trachea (a) neither CB(1) nor CB(2) cannabinoid receptor-mediated modulation of acetylcholine release occurs; (b) vanilloid VR1-like receptors appear involved in R(+)-methanandamide inhibitory activity on the cholinergic response to electrical field stimulation.

  19. Distinctive Modulation of Dopamine Release in the Nucleus Accumbens Shell Mediated by Dopamine and Acetylcholine Receptors.

    Science.gov (United States)

    Shin, Jung Hoon; Adrover, Martin F; Alvarez, Veronica A

    2017-11-15

    Nucleus accumbens (NAc) shell shows unique dopamine (DA) signals in vivo and plays a unique role in DA-dependent behaviors such as reward-motivated learning and the response to drugs of abuse. A disynaptic mechanism for DA release was reported and shown to require synchronized firing of cholinergic interneurons (CINs) and activation of nicotinic acetylcholine (ACh) receptors (nAChRs) in DA neuron (DAN) axons. The properties of this disynaptic mechanism of DA transmission are not well understood in the NAc shell. In this study, in vitro fast-scan cyclic voltammetry was used to examine the modulation of DA transmission evoked by CINs firing in the shell of mice and compared with other striatal regions. We found that DA signals in the shell displayed significant degree of summation in response to train stimulation of CINs, contrary to core and dorsal striatum. The summation was amplified by a D2-like receptor antagonist and experiments with mice with targeted deletion of D2 receptors to DANs or CINs revealed that D2 receptors in CINs mediate a fast inhibition observed within 100 ms of the first pulse, whereas D2 autoreceptors in DAN terminals are engaged in a slower inhibition that peaks at ∼500 ms. ACh also contributes to the use-dependent inhibition of DA release through muscarinic receptors only in the shell, where higher activity of acetylcholinesterase minimizes nAChR desensitization and promotes summation. These findings show that DA signals are modulated differentially by endogenous DA and ACh in the shell, which may underlie the unique features of shell DA signals in vivo SIGNIFICANCE STATEMENT The present study reports that dopamine (DA) release evoked by activation of cholinergic interneurons displays a high degree of summation in the shell and shows unique modulation by endogenous DA and acetylcholine. Desensitization of nicotinic receptors, which is a prevailing mechanism for use-dependent inhibition in the nucleus accumbens core and dorsal striatum, is

  20. [Role of calcium ions in the mechanism of action of acetylcholine on energy metabolism in rat liver mitochondria].

    Science.gov (United States)

    Vatamaniuk, M Z; Artym, V V; Kuka, O B; Doliba, M M; Shostakovs'ka, I V

    1996-01-01

    It is shown that administration of acetylcholine to animals (50 micrograms per 100 g of body weight) leads to the activation of respiration and oxidative phosphorylation in the rat liver mitochondria under oxidation of alpha-ketoglutarate; this effect depends on the concentration of calcium ions in the incubation medium of mitochondria. The rate of ADP-stimulated respiration of mitochondria of experimental animals reaches its maximum level under lower concentrations of Ca2+ than in the control animals. The results of investigation of dependence of acetyl choline effect on respiration of mitochondria on the concentration of alpha-ketoglutarate in calcium and calcium-free incubation medium have shown that the half-maximum effect of acetylcholine is observed in calcium medium at lower concentration of the substrate than in calcium-free medium. The latter indicates to the increase of affinity of alpha-ketoglutarate dehydrogenase to alpha-ketoglutarate under these conditions. It is found out that acetylcholine (1.10(-8) M) increases the rate of ADP- and Ca(2+)-stimulated respiration of mitochondria of isolated perfused rat liver, while mutual effect of verapamyl and niphedipin removes this effect.

  1. Simultaneous improvement in production of microalgal biodiesel and high-value alpha-linolenic acid by a single regulator acetylcholine.

    Science.gov (United States)

    Parsaeimehr, Ali; Sun, Zhilan; Dou, Xiao; Chen, Yi-Feng

    2015-01-01

    Photoautotrophic microalgae are a promising avenue for sustained biodiesel production, but are compromised by low yields of biomass and lipids at present. We are developing a chemical approach to improve microalgal accumulation of feedstock lipids as well as high-value alpha-linolenic acid which in turn might provide a driving force for biodiesel production. We demonstrate the effectiveness of the small bioactive molecule "acetylcholine" on accumulation of biomass, total lipids, and alpha-linolenic acid in Chlorella sorokiniana. The effectiveness exists in different species of Chlorella. Moreover, the precursor and analogs of acetylcholine display increased effectiveness at higher applied doses, with maximal increases by 126, 80, and 60% over controls for biomass, total lipids, and alpha-linolenic acid, respectively. Production of calculated biodiesel was also improved by the precursor and analogs of acetylcholine. The biodiesel quality affected by changes in microalgal fatty acid composition was addressed. The chemical approach described here could improve the lipid yield and biodiesel production of photoautotrophic microalgae if combined with current genetic approaches.

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

    Science.gov (United States)

    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.

  3. Studies of two naturally occurring compounds which effect release of acetylcholine from synaptosomes

    International Nuclear Information System (INIS)

    Koenig, M.L.

    1985-01-01

    Two naturally occurring compounds which effect the release of neurotransmitter from synaptosomes have been purified to apparent homogeneity. Iotrochotin (IOT) isolated from wound exudate of the Caribbean purple bleeder sponge promotes release in a manner that is independent of the extracellular Ca 2+ ion concentration. Leptinotarsin (LPT-d), a protein taken from hemolymph of the Colorado potato beetle, Leptinotarsa decemlineata, stimulates Ca 2+ -dependent release. IOT is slightly acidic and has a molecular weight of approximately 18 kD. [ 3 H]acetylcholine which has been introduced into synaptosomes as [ 3 H]choline can be released by IOT. The toxin releasable pool of labelled neurotransmitter is not depleted by depolarization of the synaptosomes with high potassium, and therefore seems to be primarily extravesicular. LPT-d is a larger protein (molecular weight = 45 kD) than IOT, and seems to effect primarily vesicular release by opening at least one type of presynaptic Ca 2+ channel. The facilitatory effects of the toxin on synaptosomal release can be inhibited by inorganic Ca 2+ channel antagonists, but are not generally affected by organic antagonists

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Segregation of lipids near acetylcholine-receptor channels imaged by cryo-EM

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    Nigel Unwin

    2017-07-01

    Full Text Available Rapid communication at the chemical synapse depends on the action of ion channels residing in the postsynaptic membrane. The channels open transiently upon the binding of a neurotransmitter released from the presynaptic nerve terminal, eliciting an electrical response. Membrane lipids also play a vital but poorly understood role in this process of synaptic transmission. The present study examines the lipid distribution around nicotinic acetylcholine (ACh receptors in tubular vesicles made from postsynaptic membranes of the Torpedo ray, taking advantage of the recent advances in cryo-EM. A segregated distribution of lipid molecules is found in the outer leaflet of the bilayer. Apparent cholesterol-rich patches are located in specific annular regions next to the transmembrane helices and also in a more extended `microdomain' between the apposed δ subunits of neighbouring receptors. The particular lipid distribution can be interpreted straightforwardly in relation to the gating movements revealed by an earlier time-resolved cryo-EM study, in which the membranes were exposed briefly to ACh. The results suggest that in addition to stabilizing the protein, cholesterol may play a mechanical role by conferring local rigidity to the membrane so that there is productive coupling between the extracellular and membrane domains, leading to opening of the channel.

  6. Segregation of acetylcholine and GABA in the rat superior cervical ganglia: functional correlation.

    Directory of Open Access Journals (Sweden)

    Diana eElinos

    2016-04-01

    Full Text Available Sympathetic neurons have the capability to segregate their neurotransmitters (NTs and co-transmitters to separate varicosities of single axons; furthermore, in culture, these neurons can even segregate classical transmitters. In vivo sympathetic neurons employ acetylcholine (ACh and other classical NTs such as gamma aminobutyric acid (GABA. Herein, we explore whether these neurons in vivo segregate these classical NTs in the superior cervical ganglia of the rat. We determined the topographical distribution of GABAergic varicosities, somatic GABAA receptor, as well as the regional distribution of the segregation of ACh and GABA. We evaluated possible regional differences in efficacy of ganglionic synaptic transmission, in the sensitivity of GABAA receptor to GABA and to the competitive antagonist picrotoxin (PTX. We found that sympathetic preganglionic neurons in vivo do segregate ACh and GABA. GABAergic varicosities and GABAA receptor expression showed a rostro-caudal gradient along ganglia; in contrast, segregation exhibited a caudo-rostral gradient. These uneven regional distributions in expression of GABA, GABAA receptors, and level segregation correlate with stronger synaptic transmission found in the caudal region. Accordingly, GABAA receptors of rostral region show larger sensitivity to GABA and PTX. These results suggest the presence of different types of GABAA receptors in each region that result in a different regional levels of endogenous GABA inhibition. Finally, we discuss a possible correlation of these different levels of GABA modulation and the function of the target organs innervated by rostral and caudal ganglionic neurons.

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

    International Nuclear Information System (INIS)

    Rotzler, S.; Brenner, H.R.

    1990-01-01

    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 125 I-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

  8. Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients

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    Maria Di Bari

    2016-11-01

    Full Text Available Multiple sclerosis (MS is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE and butyrylcholinesterase (BuChE. Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD, is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

  9. Drugs Interfering with Muscarinic Acetylcholine Receptors and Their Effects on Place Navigation

    Directory of Open Access Journals (Sweden)

    Jan Svoboda

    2017-11-01

    Full Text Available Muscarinic acetylcholine receptors (mAChRs have been found to regulate many diverse functions, ranging from motivation and feeding to spatial navigation, an important and widely studied type of cognitive behavior. Systemic administration of non-selective antagonists of mAChRs, such as scopolamine or atropine, have been found to have adverse effects on a vast majority of place navigation tasks. However, many of these results may be potentially confounded by disruptions of functions other than spatial learning and memory. Although studies with selective antimuscarinics point to mutually opposite effects of M1 and M2 receptors, their particular contribution to spatial cognition is still poorly understood, partly due to a lack of truly selective agents. Furthermore, constitutive knock-outs do not always support results from selective antagonists. For modeling impaired spatial cognition, the scopolamine-induced amnesia model still maintains some limited validity, but there is an apparent need for more targeted approaches such as local intracerebral administration of antagonists, as well as novel techniques such as optogenetics focused on cholinergic neurons and chemogenetics aimed at cells expressing metabotropic mAChRs.

  10. Autoradiographic localization of nicotinic acetylcholine receptors in the brain of the zebra finch (Poephila guttata)

    International Nuclear Information System (INIS)

    Watson, J.T.; Adkins-Regan, E.; Whiting, P.; Lindstrom, J.M.; Podleski, T.R.

    1988-01-01

    We have localized nicotinic acetylcholine receptors in the zebra finch brain by using three 125I-labelled ligands: alpha bungarotoxin and two monoclonal antibodies to neuronal nicotinic receptors. Unfixed brains from intact adult male and female zebra finches were prepared for in vitro autoradiography. Low-resolution film autoradiograms and high-resolution emulsion autoradiograms were prepared for each of the three ligands. The major brain structures that bind all three of the ligands are hippocampus; hyperstriatum dorsalis; hyperstriatum ventralis; nucleus lentiformis mesencephali; nucleus pretectalis, some layers of the optic tectum; nucleus mesencephalicus lateralis; pars dorsalis; locus ceruleus; and all cranial motor nuclei except nucleus nervi hypoglossi. The major structures labelled only by [125I]-alpha bungarotoxin binding included hyperstriatum accessorium and the nuclei: preopticus medialis, medialis hypothalami posterioris, semilunaris, olivarius inferior, and the periventricular organ. Of the song control nuclei, nucleus magnocellularis of the anterior neostriatum; hyperstriatum ventralis, pars caudalis; nucleus intercollicularis; and nucleus hypoglossus were labelled. The binding patterns of the two antibodies were similar to one another but not identical. Both labelled nucleus spiriformis lateralis and nucleus geniculatus lateralis, pars ventralis especially heavily and also labelled the nucleus habenula medialis; nucleus subpretectalis; nucleus isthmi, pars magnocellularis; nucleus reticularis gigantocellularis; nucleus reticularis lateralis; nucleus tractus solitarii; nucleus vestibularis dorsolateralis; nucleus vestibularis lateralis; nucleus descendens nervi trigemini; and the deep cerebellar nuclei

  11. The effect of exogenous acetylcholine and other cholinergic agents on photoperiodic flower induction of Pharbitis nil

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    Hanna Łukasiewicz-Rutkowska

    2014-01-01

    Full Text Available Exogenous acetylcholine (ACh, acetylcholinesterase (AChE inhibitors, as well as agonists and antagonists of ACh receptors applied on the cotyledons of 5-day-old seedlings of Pharbitis nil during a 16-hour long inductive night or during a 12-hour long subinductive night, do not essentially influence the flower bud formation. Also the application of above mentioned substances to the seedlings growing under the conditions of 72 hours of darkness, 24 hours of light and then 24 hours of darkness does not influence in an essential way flowering of P. nil. On the other hand, applying these substances on the cotyledons of P. nil during 24-hour-long inductive night, preceded by 72 hours of darkness and then 24 hours of light of lowered intensity finished by 15-minute-long impulse of far red light which inhibit flowering, caused the reversion of the effect of far red light irradiation and stimulated the flowering. The obtained results suggest that endogenous system ACh/AChE could participate in the mechanism of a phytochrome controlled flowering of short day plants.

  12. A New Role for Attentional Corticopetal Acetylcholine in Cortical Memory Dynamics

    Science.gov (United States)

    Fujii, Hiroshi; Kanamaru, Takashi; Aihara, Kazuyuki; Tsuda, Ichiro

    2011-09-01

    Although the role of corticopetal acetylcholine (ACh) in higher cognitive functions is increasingly recognized, the questions as (1) how ACh works in attention(s), memory dynamics and cortical state transitions, and also (2) why and how loss of ACh is involved in dysfunctions such as visual hallucinations in dementia with Lewy bodies and deficit of attention(s), are not well understood. From the perspective of a dynamical systems viewpoint, we hypothesize that transient ACh released under top-down attention serves to temporarily invoke attractor-like memories, while a background level of ACh reverses this process returning the dynamical nature of the memory structure back to attractor ruins (quasi-attractors). In fact, transient ACh loosens inhibitions of py ramidal neurons (PYRs) by P V+ fas t spiking (FS) i nterneurons, while a baseline ACh recovers inhibitory actions of P V+ FS. Attentional A Ch thus dynamically modifies brain's connectivity. Th e core of this process is in the depression of GABAergic inhibitory currents in PYRs due to muscarinic (probably M2 subtype) presyn aptic effects on GABAergic synapses of PV+ FS neurons

  13. Functional interaction of nicotinic acetylcholine receptors and Na+/K+ ATPase from Locusta migratoria manilensis (Meyen).

    Science.gov (United States)

    Bao, Haibo; Sun, Huahua; Xiao, Youxin; Zhang, Yixi; Wang, Xin; Xu, Xiaoyong; Liu, Zewen; Fang, Jichao; Li, Zhong

    2015-03-06

    Associated proteins are important for the correct functioning of nicotinic acetylcholine receptors (nAChRs). In the present study, a neonicotinoid-agarose affinity column was used to isolate related proteins from a solubilized membrane preparation from the nervous system of Locusta migratoria manilensis (Meyen). 1530 peptides were identified and most of them were involved in the membranous structure, molecular interaction and cellular communication. Among these peptides, Na(+)/K(+) ATPase had the highest MASCOT score and were involved in the molecular interaction, which suggested that Na(+)/K(+) ATPase and nAChRs might have strong and stable interactions in insect central nervous system. In the present study, functional interactions between nAChRs and Na(+)/K(+) ATPase were examined by heterologous expression in Xenopus oocytes. The results showed that the activated nAChRs increased pump currents of Na(+)/K(+) ATPase, which did not require current flow through open nAChRs. In turn, Na(+)/K(+) ATPase significantly increased agonist sensitivities of nAChRs in a pump activity-independent manner and reduced the maximum current (Imax) of nAChRs. These findings provide novel insights concerning the functional interactions between insect nAChRs and Na(+)/K(+) ATPase.

  14. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    Science.gov (United States)

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  15. Optical acetylcholine sensor based on free base porphyrin as a chromoionophore.

    Science.gov (United States)

    Mroczkiewicz, Monika; Pietrzak, Mariusz; Górski, Łukasz; Malinowska, Elżbieta

    2011-09-21

    In this work, the possibility of application of free base porphyrin as a lipophilic pH chromoionophore for the preparation of optical cation-selective sensors was investigated. The properties of polymeric membranes, containing porphyrins of different structures, namely tetraphenylporphyrin (TPP) and octaethylporphyrin (OEP), were compared. Changes in equilibrium between protonated and deprotonated form of porphyrin, resulting from variations in ACh concentration, were evaluated. The influence of various factors (kind and quantity of anionic additive and porphyrin in the membrane phase, pH of sample solution) on initial equilibrium was studied. The best membrane composition was chosen as: TPP 3 wt.%, KTFPB 175 mol.% relative to ionophore, PVC:o-NPOE (1 : 4) and measuring buffer solution: 0.05 M MES, pH 4.5. Selectivity, response stability, reversibility and repeatability tests were carried out for chosen sensor. Developed sensor allowed for the determination of a model analyte, acetylcholine, at the concentration range of 10(-5) to 10(-2) M, both in stationary and flow-injection system. Sensor response was reversible and repeatable in the mentioned concentration range.

  16. Selective decreases of nicotinic acetylcholine receptors in PC12 cells exposed to fluoride

    International Nuclear Information System (INIS)

    Chen Jia; Shan, K.-R.; Long, Y.-G.; Wang, Y.-N.; Nordberg, Agneta; Guan, Z.-Z.

    2003-01-01

    In an attempt to elucidate the mechanism by which excessive fluoride damages the central nervous system, the effects of exposure of PC12 cells to different concentrations of fluoride for 48 h on nicotinic acetylcholine receptors (nAChRs) were characterized here. Significant reductions in the number of binding sites for both [ 3 H]epibatidine and [ 125 I]α-bungarotoxin, as well as a significant decrease in the B max value for the high-affinity of epibatidine binding site were observed in PC12 cells subjected to high levels of fluoride. On the protein level, the α3 and α7 subunits of nAChRs were also significantly decreased in the cells exposed to high concentrations of fluoride. In contrast, such exposure had no significant effect on the level of the β2 subunit. These findings suggest that selective decreases in the number of nAChRs may play an important role in the mechanism(s) by which fluoride causes dysfunction of the central nervous system

  17. Acetylcholine release in the hippocampus during the operant conditioned reflex and the footshock stimulus in rats.

    Science.gov (United States)

    Dong, Yu; Mao, Jianjun; Shangguan, Dihua; Zhao, Rui; Liu, Guoquan

    2004-10-14

    The activity of the septo-hippocampal cholinergic pathway was investigated by measuring changes in the extracellular acetylcholine (ACh) levels in the hippocampus, by means of microdialysis, during the operant conditioned reflex and the repeated footshock stimulus. Microdialysis samplings were conducted in a Skinner box where lights were delivered as conditioned stimuli (CS) paired with footshocks as unconditioned stimuli (US). Two groups of rats were used. Extracellular ACh and choline (Ch) in samples collected at 6min intervals were assessed by high-performance liquid chromatography with electrochemical detection. The elevation of hippocampus ACh was observed in the two experimental groups. The increase in ACh during aversive stimulus (footshock) was significantly larger and was probably related to the number of footshocks. There might be moderate increase in the hippocampal ACh release during the retrieval of information. The concentration of choline showed no significant fluctuation in the two groups during the whole process. This experiment explored in more detail hippocampal cholinergic activity in relation to the two different procedures.

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

    Science.gov (United States)

    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.

  19. Pathogenesis of Abdominal Aortic Aneurysms: Role of Nicotine and Nicotinic Acetylcholine Receptors

    Directory of Open Access Journals (Sweden)

    Zong-Zhuang Li

    2012-01-01

    Full Text Available Inflammation, proteolysis, smooth muscle cell apoptosis, and angiogenesis have been implicated in the pathogenesis of abdominal aortic aneurysms (AAAs, although the well-defined initiating mechanism is not fully understood. Matrix metalloproteinases (MMPs such as MMP-2 and -9 and other proteinases degrading elastin and extracellular matrix are the critical pathogenesis of AAAs. Among the risk factors of AAAs, cigarette smoking is an irrefutable one. Cigarette smoke is practically involved in various aspects of the AAA pathogenesis. Nicotine, a major alkaloid in tobacco leaves and a primary component in cigarette smoke, can stimulate the MMPs expression by vascular SMCs, endothelial cells, and inflammatory cells in vascular wall and induce angiogenesis in the aneurysmal tissues. However, for the inflammatory and apoptotic processes in the pathogenesis of AAAs, nicotine seems to be moving in just the opposite direction. Additionally, the effects of nicotine are probably dose dependent or associated with the exposure duration and may be partly exerted by its receptors—nicotinic acetylcholine receptors (nAChRs. In this paper, we will mainly discuss the pathogenesis of AAAs involving inflammation, proteolysis, smooth muscle cell apoptosis and angiogenesis, and the roles of nicotine and nAChRs.

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

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    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.

  1. Acetylcholine promotes the emergence and elongation of lateral roots of Raphanus sativus.

    Science.gov (United States)

    Sugiyama, Kou-ichi; Tezuka, Takafumi

    2011-10-01

    Radish (Raphanus sativus L.) was grown on four layers of paper towel moistened with distilled water with and without acetylcholine (ACh) for five days in the dark after sowing. ACh at 1 nM promoted the growth (emergence and elongation) of lateral roots of radish plants, but had no effect on the stems and main roots. Moreover, ACh enhanced the dry weight of roots [main (primary) + lateral roots]. Neostigmine, an inhibitor of acetylcholinesterase (AChE) also promoted the emergence and elongation of lateral roots, and atropine, a competitive inhibitor of ACh receptor, suppressed the emergence and elongation. ACh suppressed the activity of AChE and increased the amount of proteins and pyridine nucleotides (NAD and NADH) in the roots of the seedlings. It also increased the activities of NAD-forming enzymes [NAD synthetase and ATP-nicotinamide mononucleotide (ATP-NMN) adenyltransferase], and enhanced the amount of DNA in the roots of the seedlings. The relationship between ACh and the emergence and growth of lateral roots was discussed from a biochemical viewpoint.

  2. Integrated regulation of AMPA glutamate receptor phosphorylation in the striatum by dopamine and acetylcholine.

    Science.gov (United States)

    Xue, Bing; Chen, Elton C; He, Nan; Jin, Dao-Zhong; Mao, Li-Min; Wang, John Q

    2017-01-01

    Dopamine (DA) and acetylcholine (ACh) signals converge onto protein kinase A (PKA) in medium spiny neurons of the striatum to control cellular and synaptic activities of these neurons, although underlying molecular mechanisms are less clear. Here we measured phosphorylation of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) at a PKA site (S845) as an indicator of AMPAR responses in adult rat brains in vivo to explore how DA and ACh interact to modulate AMPARs. We found that subtype-selective activation of DA D1 receptors (D1Rs), D2 receptors (D2Rs), or muscarinic M4 receptors (M4Rs) induced specific patterns of GluA1 S845 responses in the striatum. These defined patterns support a local multitransmitter interaction model in which D2Rs inhibited an intrinsic inhibitory element mediated by M4Rs to enhance the D1R efficacy in modulating AMPARs. Consistent with this, selective enhancement of M4R activity by a positive allosteric modulator resumed the cholinergic inhibition of D1Rs. In addition, D1R and D2R coactivation recruited GluA1 and PKA preferentially to extrasynaptic sites. In sum, our in vivo data support an existence of a dynamic DA-ACh balance in the striatum which actively modulates GluA1 AMPAR phosphorylation and trafficking. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line

    International Nuclear Information System (INIS)

    Blusztajn, J.K.; Liscovitch, M.; Richardson, U.I.

    1987-01-01

    Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used a precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here the authors used a population of purely cholinergic cells (human neuroblastomas, LA-N-2), incubated in the presence of [methyl- 3 H]methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. Three peaks of radioactive material that cochromatographed with authentic AcCho, choline, and phosphocholine were observed when the water-soluble metabolites of the [ 3 H]PtdCho were purified by high-performance liquid chromatography. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine

  4. Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein.

    Science.gov (United States)

    Bertaccini, Edward J; Lindahl, Erik; Sixma, Titia; Trudell, James R

    2008-04-01

    Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Bilirubin Modulates Acetylcholine Receptors In Rat Superior Cervical Ganglionic Neurons In a Bidirectional Manner

    Science.gov (United States)

    Zhang, Chengmi; Wang, Zhenmeng; Dong, Jing; Pan, Ruirui; Qiu, Haibo; Zhang, Jinmin; Zhang, Peng; Zheng, Jijian; Yu, Weifeng

    2014-01-01

    Autonomic dysfunction as a partial contributing factor to cardiovascular instability in jaundiced patients is often associated with increased serum bilirubin levels. Whether increased serum bilirubin levels could directly inhibit sympathetic ganglion transmission by blocking neuronal nicotinic acetylcholine receptors (nAChRs) remains to be elucidated. Conventional patch-clamp recordings were used to study the effect of bilirubin on nAChRs currents from enzymatically dissociated rat superior cervical ganglia (SCG) neurons. The results showed that low concnetrations (0.5 and 2 μM) of bilirubin enhanced the peak ACh-evoked currents, while high concentrations (3 to 5.5 µM) of bilirubin suppressed the currents with an IC50 of 4 ± 0.5 μM. In addition, bilirubin decreased the extent of desensitization of nAChRs in a concentration-dependent manner. This inhibitory effect of bilirubin on nAChRs channel currents was non-competitive and voltage independent. Bilirubin partly improved the inhibitory effect of forskolin on ACh-induced currents without affecting the action of H-89. These data suggest that the dual effects of enhancement and suppression of bilirubin on nAChR function may be ascribed to the action mechanism of positive allosteric modulation and direct blockade. Thus, suppression of sympathetic ganglionic transmission through postganglionic nAChRs inhibition may partially contribute to the adverse cardiovascular effects in jaundiced patients. PMID:25503810

  7. Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis.

    Science.gov (United States)

    Lennon, V A; Lambert, E H; Leiby, K R; Okarma, T B; Talib, S

    1991-04-01

    A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.

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

    Energy Technology Data Exchange (ETDEWEB)

    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}.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    Gu, Y.

    1989-01-01

    The specificity of the antibodies in the serum of a patient with myasthenia gravis for a the α-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 22 Na uptake into C2 myotubes as a measured of the receptor function. Antibodies specific for the γ, δ, and ε 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 (α, β, γ, and δ) subunits of mammalian muscle AChRs on immunoblots. When AChRs from embryonic, neonatal, normal and denervated adult muscles were compared on immunoblots, the α, β, and δ subunits were identical in all four receptor preparations, with or without endoglycosidase digestion. The spatial and temporal distribution of the γ- and ε- 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 γ is replaced by ε

  11. Unraveling a molecular determinant for clathrin-independent internalization of the M2 muscarinic acetylcholine receptor

    Science.gov (United States)

    Wan, Min; Zhang, Wenhua; Tian, Yangli; Xu, Chanjuan; Xu, Tao; Liu, Jianfeng; Zhang, Rongying

    2015-01-01

    Endocytosis and postendocytic sorting of G-protein-coupled receptors (GPCRs) is important for the regulation of both their cell surface density and signaling profile. Unlike the mechanisms of clathrin-dependent endocytosis (CDE), the mechanisms underlying the control of GPCR signaling by clathrin-independent endocytosis (CIE) remain largely unknown. Among the muscarinic acetylcholine receptors (mAChRs), the M4 mAChR undergoes CDE and recycling, whereas the M2 mAChR is internalized through CIE and targeted to lysosomes. Here we investigated the endocytosis and postendocytic trafficking of M2 mAChR based on a comparative analysis of the third cytoplasmic domain in M2 and M4 mAChRs. For the first time, we identified that the sequence 374KKKPPPS380 servers as a sorting signal for the clathrin-independent internalization of M2 mAChR. Switching 374KKKPPPS380 to the i3 loop of the M4 mAChR shifted the receptor into lysosomes through the CIE pathway; and therefore away from CDE and recycling. We also found another previously unidentified sequence that guides CDE of the M2 mAChR, 361VARKIVKMTKQPA373, which is normally masked in the presence of the downstream sequence 374KKKPPPS380. Taken together, our data indicate that endocytosis and postendocytic sorting of GPCRs that undergo CIE could be sequence-dependent. PMID:26094760

  12. Acetylcholine esterase activity in mild cognitive impairment and Alzheimer's disease

    Energy Technology Data Exchange (ETDEWEB)

    Herholz, Karl [University of Manchester, Wolfson Molecular Imaging Centre, Clinical Neuroscience, Manchester (United Kingdom); University of Cologne, Cologne (Germany)

    2008-03-15

    Impairment of cholinergic neurotransmission is a well-established fact in Alzheimer's disease (AD), but there is controversy about its relevance at the early stages of the disease and in mild cognitive impairment (MCI). In vivo positron emission tomography imaging of cortical acetylcholine esterase (AChE) activity as a marker of cholinergic innervation that is expressed by cholinergic axons and cholinoceptive neurons has demonstrated a reduction of this enzyme activity in manifest AD. The technique is also useful to measure the inhibition of cerebral AChE induced by cholinesterase inhibitors for treatment of dementia symptoms. A reduction of cortical AchE activity was found consistently in all studies of AD and in few cases of MCI who later concerted to AD. The in vivo findings in MCI and very mild AD are still preliminary, and studies seem to suggest that cholinergic innervation and AChE as the main degrading enzyme are both reduced, which might result in partial compensation of their effect. (orig.)

  13. Neuronal M3 muscarinic acetylcholine receptors are essential for somatotroph proliferation and normal somatic growth.

    Science.gov (United States)

    Gautam, Dinesh; Jeon, Jongrye; Starost, Matthew F; Han, Sung-Jun; Hamdan, Fadi F; Cui, Yinghong; Parlow, Albert F; Gavrilova, Oksana; Szalayova, Ildiko; Mezey, Eva; Wess, Jürgen

    2009-04-14

    The molecular pathways that promote the proliferation and maintenance of pituitary somatotrophs and other cell types of the anterior pituitary gland are not well understood at present. However, such knowledge is likely to lead to the development of novel drugs useful for the treatment of various human growth disorders. Although muscarinic cholinergic pathways have been implicated in regulating somatotroph function, the physiological relevance of this effect and the localization and nature of the receptor subtypes involved in this activity remain unclear. We report the surprising observation that mutant mice that selectively lack the M(3) muscarinic acetylcholine receptor subtype in the brain (neurons and glial cells; Br-M3-KO mice) showed a dwarf phenotype associated with a pronounced hypoplasia of the anterior pituitary gland and a marked decrease in pituitary and serum growth hormone (GH) and prolactin. Remarkably, treatment of Br-M3-KO mice with CJC-1295, a synthetic GH-releasing hormone (GHRH) analog, rescued the growth deficit displayed by Br-M3-KO mice by restoring normal pituitary size and normal serum GH and IGF-1 levels. These findings, together with results from M(3) receptor/GHRH colocalization studies and hypothalamic hormone measurements, support a model in which central (hypothalamic) M(3) receptors are required for the proper function of hypothalamic GHRH neurons. Our data reveal an unexpected and critical role for central M(3) receptors in regulating longitudinal growth by promoting the proliferation of pituitary somatotroph cells.

  14. Efficient Expression of Acetylcholine-Binding Protein from Aplysia californica in Bac-to-Bac System

    Directory of Open Access Journals (Sweden)

    Bo Lin

    2014-01-01

    Full Text Available The Bac-to-Bac baculovirus expression system can efficiently produce recombinant proteins, but the system may have to be optimized to achieve high-level expression for different candidate proteins. We reported here the efficient expression of acetylcholine-binding proteins from sea hares Aplysia californica (Ac-AChBP and a convenient method to monitor protein expression level in this expression system. Three key factors affecting expression of Ac-AChBP were optimized for maximizing the yield, which included the cell density, volume of the infecting baculovirus inoculums, and the culturing time of postinfection. We have found it to reach a high yield of ∼5 mg/L, which needs 55 h incubation after infection at the cell density of 2 × 106 cells/mL with an inoculum volume ratio of 1 : 100. The optimized expression system in this study was also applied for expressing another protein Ls-AChBP from Lymnaea stagnalis successfully. Therefore, this established method is helpful to produce high yields of AChBP proteins for X-ray crystallographic structural and functional studies.

  15. BDNF Up-Regulates α7 Nicotinic Acetylcholine Receptor Levels on Subpopulations of Hippocampal Interneurons

    Science.gov (United States)

    Massey, Kerri A.; Zago, Wagner M.; Berg, Darwin K.

    2006-01-01

    In the hippocampus, brain-derived neurotrophic factor (BDNF) regulates a number of synaptic components. Among these are nicotinic acetylcholine receptors containing α7 subunits (α7-nAChRs), which are interesting because of their relative abundance in the hippocampus and their high relative calcium permeability. We show here that BDNF elevates surface and intracellular pools of α7-nAChRs on cultured hippocampal neurons and that glutamatergic activity is both necessary and sufficient for the effect. Blocking transmission through NMDA receptors with APV blocked the BDNF effect; increasing spontaneous excitatory activity with the GABAA receptor antagonist bicuculline replicated the BDNF effect. BDNF antibodies blocked the BDNF-mediated increase but not the bicuculline one, consistent with enhanced glutamatergic activity acting downstream from BDNF. Increased α7-nAChR clusters were most prominent on interneuron subtypes known to innervate directly excitatory neurons. The results suggest that BDNF, acting through glutamatergic transmission, can modulate hippocampal output in part by controlling α7-nAChR levels. PMID:17029981

  16. Dopamine regulation of [3H]acetylcholine release from guinea-pig stomach

    International Nuclear Information System (INIS)

    Kusunoki, M.; Taniyama, K.; Tanaka, C.

    1985-01-01

    The involvement of dopamine receptors in cholinergic transmission of guinea-pig stomach was investigated by analyzing the effects of dopamine receptor agonists and antagonists on acetylcholine (ACh) release from this organ. Electrical stimulation (1-20 Hz) of strips of guinea-pig stomach preloaded with [ 3 H] choline induced a [ 3 H]ACh release that was calcium dependent and tetrodotoxin sensitive. Dopamine inhibited this transmural stimulation-induced [ 3 H]ACh release in a concentration-dependent manner (10(-8)-10(-4) M). This effect of dopamine was not altered by 10(-5) M hexamethonium, thereby suggesting that the major dopamine receptors are located on the postganglionic cholinergic neurons. Concentration-response curves for dopamine on [ 3 H]ACh release were inhibited by haloperidol, sulpiride and domperidone but not by prazosin, yohimbine, propranolol and ketanserin. LY 171555, an agonist for the D2 dopamine receptor, but not SKF 38-393, an agonist for the D1 dopamine receptor, to some extent decreased the release of [ 3 H]ACh induced by transmural stimulation. In view of the results, the release of ACh from postganglionic cholinergic neurons is probably required through dopamine receptors antagonized by D2 antagonists but not by adrenergic or serotonin receptor antagonists

  17. Action of hypo- and hyperthyroidism on the postmortal decay of acetylcholine in the rat spinal cord.

    Science.gov (United States)

    Molinengo, L; Cassone, M C; Oggero, L

    1986-01-01

    The postmortal decay of acetylcholine (Ach) was studied in the cervical spinal cords of rats in conditions of hyper- and hypothyroidism. The modifications of thyroid function were achieved either by chronic (20-25 days) administration of l-thyroxine or of methimazole. The basal metabolic rate and plasma T4 concentration were measured to estimate the degree of modification of thyroid activity. The levels of Ach at the start of postmortal decay were evaluated by extrapolation to time 0 of the curves of the postmortal decay of Ach and the levels of Ach at stabilization were estimated from the means of all the measures made at lapses of time over 100-200 s from death. In low and high hypothyroidism a reduction (53 and 72%, respectively) of the levels of Ach was found. A similar effect was found in hyperthyroidism: a 73 and 63% reduction of Ach levels in high and low hyperthyroidism, respectively. The level of Ach at stabilization of the postmortal decay increased only in hyperthyroid rats. The process by which Ach is destroyed is not modified in hyper- or hypothyroidism.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  19. The therapeutic potential of nicotinic acetylcholine receptor agonists for pain control.

    Science.gov (United States)

    Decker, M W; Meyer, M D; Sullivan, J P

    2001-10-01

    Due to the limitations of currently available analgesics, a number of novel alternatives are currently under investigation, including neuronal nicotinic acetylcholine receptor (nAChR) agonists. During the 1990s, the discovery of the antinociceptive properties of the potent nAChR agonist epibatidine in rodents sparked interest in the analgesic potential of this class of compounds. Although epibatidine also has several mechanism-related toxicities, the identification of considerable nAChR diversity suggested that the toxicities and therapeutic actions of the compound might be mediated by distinct receptor subtypes. Consistent with this view, a number of novel nAChR agonists with antinociceptive activity and improved safety profiles in preclinical models have now been identified, including A-85380, ABT-594, DBO-83, SIB-1663 and RJR-2403. Of these, ABT-594 is the most advanced and is currently in Phase II clinical evaluation. Nicotinically-mediated antinociception has been demonstrated in a variety of rodent pain models and is likely mediated by the activation of descending inhibitory pathways originating in the brainstem with the predominant high-affinity nicotine site in brain, the alpha4beta2 subtype, playing a critical role. Thus, preclinical findings suggest that nAChR agonists have the potential to be highly efficacious treatments in a variety of pain states. However, clinical proof-of-principle studies will be required to determine if nAChR agonists are active in pathological pain.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. Action of nereistoxin on recombinant neuronal nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes.

    Science.gov (United States)

    Raymond Delpech, Valérie; Ihara, Makoto; Coddou, Claudio; Matsuda, Kazuhiko; Sattelle, David B

    2003-11-01

    Nereistoxin (NTX), a natural neurotoxin from the salivary glands of the marine annelid worm Lumbriconereis heteropoda, is highly toxic to insects. Its synthetic analogue, Cartap, was the first commercial insecticide based on a natural product. We have used voltage-clamp electrophysiology to compare the actions of NTX on recombinant nicotinic acetylcholine receptors (nicotinic AChRs) expressed in Xenopus laevis oocytes following nuclear injection of cDNAs. The recombinant nicotinic AChRs investigated were chicken alpha7, chicken alpha4beta2 and the Drosophila melanogaster/chicken hybrid receptors SAD/beta2 and ALS/beta2. No agonist action of NTX (0.1-100 microM) was observed on chicken alpha7, chicken alpha4beta2 and the Drosophila/chicken hybrid nicotinic AChRs. Currents elicited by ACh were reduced in amplitude by NTX in a dose-dependent manner. The toxin was slightly more potent on recombinant Drosophila/vertebrate hybrid receptors than on vertebrate homomeric (alpha7) or heteromeric (alpha4beta2) nicotinic AChRs. Block by NTX of the chicken alpha7, chicken alpha4beta2 and the SAD/beta2 and ALS/beta2 Drosophila/chicken hybrid receptors is in all cases non-competitive. Thus, the site of action on nicotinic AChRs of NTX, to which the insecticide Cartap is metabolised in insects, differs from that of the major nicotinic AChR-active insecticide, imidacloprid.

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

    International Nuclear Information System (INIS)

    Liu Yuxia; Liu Ning; Yang Yuanyou; Zan Liangbiao; Liao Jiali; Jin Jiannan; Sichuan Univ., Chengdu

    2006-01-01

    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 125/123 I. The whole procedure for radiosynthesis needs 50-55 min and more than 30% of the 125 I are found in the purified 5-[ 125 I]-A-85380. Even staying for 3 days at room temperature in vitro, the purified 5-[ 125 I]-I-85380 can maintain its stability, with a radiochemical purity of more than 95%. (authors)

  3. [3H]acetylcholine synthesis in cultured ciliary ganglion neurons: effects of myotube membranes

    International Nuclear Information System (INIS)

    Gray, D.B.; Tuttle, J.B.

    1987-01-01

    Avian ciliary ganglion neurons in cell culture were examined for the capacity to synthesize acetylcholine (ACh) from the exogenously supplied precursor, choline. Relevant kinetic parameters of the ACh synthetic system in cultured neurons were found to be virtually the same as those of the ganglionic terminals in the intact iris. Neurons were cultured in the presence of and allowed to innervate pectoral muscle; this results in an capacity for ACh synthesis. In particular, the ability to increase ACh synthesis upon demand after stimulation is affected by interaction with the target. This effect is shown to be an acceleration of the maturation of the cultured neurons. Lysed and washed membrane remnants of the muscle target were able to duplicate, in part, this effect of live target tissue on neuronal transmitter metabolism. Culture medium conditioned by muscle, and by the membrane remnants of muscle, was without significant effect. Thus, substances secreted into the medium do not play a major role in this interaction. Neurons cultured with either muscle or muscle membrane remnants formed large, elongate structures on the target membrane surface. These were not seen in the absence of the target at the times examined. This morphological difference in terminal-like structures may parallel the developmental increases in size and vesicular content of ciliary ganglion nerve terminals in the chick iris, and may relate to the increased ACh synthetic activity. The results suggest that direct contact with an appropriate target membrane has a profound, retrograde influence upon neuronal metabolic and morphological maturation

  4. Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum

    Directory of Open Access Journals (Sweden)

    Melanie Abongwa

    2018-04-01

    Full Text Available Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 ± 3.1 nM and 0.2 ± 2.3 μM. We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes. Keywords: Monepantel, Zolvix®, Nicotinic acetylcholine

  5. Stabilization of acetylcholine receptors at the neuromuscular synapse: the role of the nerve.

    Science.gov (United States)

    Ramsay, D A; Drachman, D B; Drachman, R J; Stanley, E F

    1992-05-29

    The majority of acetylcholine receptors (AChRs) at innervated neuromuscular junctions (NMJs) are stable, with half-lives averaging about 11 days in rodent muscles. In addition to the stable AChRs, approximately 18% of AChRs at these innervated junctions are rapidly turned over (RTOs), with half lives of less than 24 h. We have postulated that RTOs may be precursors of stable AChRs, and that the motor nerve may influence their stabilization. This hypothesis was tested by: (i) labeling AChRs in mouse sternomastoid (SM) muscles with 125I-alpha-BuTx; (ii) denervating one SM muscle in each mouse, and (iii) following the fate of the labeled AChRs through a 5-day period when RTOs were either stabilized or degraded. The hypothesis predicts that denervation should preclude stabilization of RTOs, resulting in a deficit of stable AChRs in denervated muscles. The results showed a highly significant (P less than 0.002) deficit of stable AChRs in denervated as compared with innervated muscles. Control experiments excluded the possibility that this deficit could be attributed to independent accelerated degradation of either RTOs or pre-existing stable AChRs. The observed deficit was quantitatively consistent with the deficit predicted by a mathematical model based on interruption of stabilization following denervation. We conclude that: (i) the observed deficit after denervation of NMJs is due to failure of stabilization of pre-existing RTOs; (ii) RTOs at normally innervated NMJs are precursors of stable AChRs; (iii) stabilization occurs after the insertion of AChRs at NMJs, and (iv) motor nerves play a key role in stabilization of RTOs. The concept of receptor stabilization has important implications for understanding the biology of the neuromuscular junction and post-synaptic plasticity.

  6. Fluoxetine Alleviates Behavioral Depression while Decreasing Acetylcholine Release in the Nucleus Accumbens Shell

    Science.gov (United States)

    Chau, David T; Rada, Pedro V; Kim, Kay; Kosloff, Rebecca A; Hoebel, Bartley G

    2011-01-01

    Selective serotonin reuptake inhibitors, such as fluoxetine, have demonstrated the ability to alleviate behavioral depression in the forced swim test; however, the sites and mechanisms of their actions remain to be further elucidated. Previous studies have suggested that behavioral depression in the swim test is mediated in part by acetylcholine (ACh) stimulating the cholinergic M1 receptors in the nucleus accumbens (NAc) shell. The current study tested whether acute, local, and chronic, subcutaneous fluoxetine treatments increase escape motivation during the swim test while simultaneously lowering extracellular ACh in the NAc shell. Experiment 1: Fluoxetine (1.0 mM) infused unilaterally in the NAc shell for 40 min reduced extracellular ACh while simultaneously increasing swimming time. Experiment 2: Fluoxetine (0.2, 0.5, and 0.75 mM) infused bilaterally in the NAc shell on day 3 dose-dependently decreased immobility and increased the total escape attempts (swimming and climbing) compared with Ringer given on day 2. Experiment 3: Fluoxetine (0.5 mM) infused bilaterally in the NAc for 40 min did not affect activities in an open field. Experiment 4: Chronic systemic fluoxetine treatment decreased immobility scores and increased total escape attempt scores compared with control saline treatment. In all, 14 days after the initial swim test, basal extracellular ACh in the shell was still elevated in the saline-treated group, but not in the fluoxetine-treated group. In summary, these data suggest that one of the potential mechanisms by which fluoxetine alleviates behavioral depression in the forced swim test may be to suppress cholinergic activities in the NAc shell. PMID:21525864

  7. Mechanism of acetylcholine receptor cluster formation induced by DC electric field.

    Directory of Open Access Journals (Sweden)

    Hailong Luke Zhang

    Full Text Available BACKGROUND: The formation of acetylcholine receptor (AChR cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells. METHODOLOGY/PRINCIPAL FINDINGS: To understand its molecular mechanism, quantum dots (QDs were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering. CONCLUSIONS: These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.

  8. Intermittent hypercapnic hypoxia effects on the nicotinic acetylcholine receptors in the developing piglet hippocampus and brainstem.

    Science.gov (United States)

    Vivekanandarajah, Arunnjah; Aishah, Atqiya; Waters, Karen A; Machaalani, Rita

    2017-05-01

    This study investigated the effects of acute (1 day) vs repeated (4 days) exposure to intermittent hypercapnic hypoxia (IHH) on the immunohistochemical expression of α2, α3, α5, α7, α9 and β2 nicotinic acetylcholine receptor (nAChR) subunits in the developing piglet hippocampus and brainstem medulla, and how prior nicotine exposure alters the response to acute IHH. Five piglet groups included: 1day IHH (1D IHH, n=9), 4days IHH (4D IHH, n=8), controls exposed only to air cycles for 1day (1D Air, n=6) or 4days (4D Air, n=5), and pre-exposed to nicotine for 13days prior to 1day IHH (Nic+1D IHH, n=7). The exposure period alternated 6min of HH (8%O 2 , 7%CO 2 , balance N 2 ) and 6min of air over 48min, while controls were switched from air-to-air. Results showed that: 1. repeated IHH induces more changes in nAChR subunit expression than acute IHH in both the hippocampus and brainstem medulla, 2. In the hippocampus, α2 and β2 changed the most (increased) following IHH and the CA3, CA2 and DG were mostly affected. In the brainstem medulla, α2, α5, α9 and β2 were changed (decreased) in most nuclei with the hypoglossal and nucleus of the solitary tract being mostly affected. 3. Pre-exposure to nicotine enhanced the changes in the hippocampus but dampened those in the brainstem medulla. These findings indicate that the nAChRs (predominantly with the α2/β2 complex) are affected by IHH in critical hippocampal and brainstem nuclei during early brain development, and that pre-exposure to nicotine alters the pattern of susceptibility to IHH. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Predicted overlapping microRNA regulators of acetylcholine packaging and degradation in neuroinflammation-related disorders

    Directory of Open Access Journals (Sweden)

    Bettina eNadorp

    2014-02-01

    Full Text Available MicroRNAs (miRNAs can notably control many targets each and regulate entire cellular pathways, but whether miRNAs can regulate complete neurotransmission processes is largely unknown. Here, we report that miRNAs with complementary sequence motifs to the key genes involved in acetylcholine (ACh synthesis and/or packaging show massive overlap with those regulating ACh degradation. To address this topic, we first searched for miRNAs that could target the 3’-untranslated regions of the choline acetyltransferase (ChAT gene that controls ACh synthesis; the vesicular ACh transporter (VAChT, encoded from an intron in the ChAT gene and the ACh hydrolyzing genes acetyl- and/or butyrylcholinesterase (AChE, BChE. Intriguingly, we found that many of the miRNAs targeting these genes are primate-specific, and that changes in their levels associate with inflammation, anxiety, brain damage, cardiac, neurodegenerative or pain-related syndromes. To validate the in vivo relevance of this dual interaction, we selected the evolutionarily conserved miR-186, which targets both the stress-inducible soluble readthrough variant AChE-R and the major peripheral cholinesterase BChE. We exposed mice to predator scent stress and searched for potential associations between consequent changes in their miR-186, AChE-R and BChE levels. Both intestinal miR-186 as well as BChE and AChE-R activities were conspicuously elevated one week post-exposure, highlighting the previously unknown involvement of miR-186 and BChE in psychological stress responses. Overlapping miRNA regulation emerges from our findings as a recently evolved surveillance mechanism over cholinergic neurotransmission in health and disease; and the corresponding miRNA details and disease relevance may serve as a useful resource for studying the molecular mechanisms underlying this surveillance.

  10. A mechanism for acetylcholine receptor gating based on structure, coupling, phi, and flip.

    Science.gov (United States)

    Gupta, Shaweta; Chakraborty, Srirupa; Vij, Ridhima; Auerbach, Anthony

    2017-01-01

    Nicotinic acetylcholine receptors are allosteric proteins that generate membrane currents by isomerizing ("gating") between resting and active conformations under the influence of neurotransmitters. Here, to explore the mechanisms that link the transmitter-binding sites (TBSs) with the distant gate, we use mutant cycle analyses to measure coupling between residue pairs, phi value analyses to sequence domain rearrangements, and current simulations to reproduce a microsecond shut component ("flip") apparent in single-channel recordings. Significant interactions between amino acids separated by >15 Å are rare; an exception is between the αM2-M3 linkers and the TBSs that are ∼30 Å apart. Linker residues also make significant, local interactions within and between subunits. Phi value analyses indicate that without agonists, the linker is the first region in the protein to reach the gating transition state. Together, the phi pattern and flip component suggest that a complete, resting↔active allosteric transition involves passage through four brief intermediate states, with brief shut events arising from sojourns in all or a subset. We derive energy landscapes for gating with and without agonists, and propose a structure-based model in which resting→active starts with spontaneous rearrangements of the M2-M3 linkers and TBSs. These conformational changes stabilize a twisted extracellular domain to promote transmembrane helix tilting, gate dilation, and the formation of a "bubble" that collapses to initiate ion conduction. The energy landscapes suggest that twisting is the most energetically unfavorable step in the resting→active conformational change and that the rate-limiting step in the reverse process is bubble formation. © 2017 Gupta et al.

  11. Analysis of the negative inotropic effect of acetylcholine on frog atrial fibres.

    Science.gov (United States)

    Nargeot, J; Garnier, D; Rougier, O

    1981-03-01

    Voltage-clamp experiments have been performed on frog atrial preparations in order to study the mechanism of the inotropic effect of acetylcholine (ACh) at various concentrations. The amplitude of the slow inward current (Is) is reduced even at low ACh concentrations; such low concentrations have little or no effect on potassium permeability. Dose-effect relationships for Is inhibition (Is/Is max) by ACh show a half amplitude dose (K0.5 around 8 X 10(-8) M ACh. The reduction of Is is attributed largely to a decrease of the maximal conductance of the slow channel (gs). Steady-state activation and inactivation parameters are not affected by ACh. Experiments in a Na-free solution (Na replaced by Li ions) or in a Ca-free solution (with EGTA) indicate that the "slow sodium current" is more sensitive to ACh than the "slow Ca current", although these two currents both seem to flow through the slow channel. The decrease of the phasic component of contraction observed in the presence of ACh is very well correlated with the decrease of Is (K0.5 = 8 X 10(-8) M ACh), while the increase of the tonic tension may be related to the outward potassium current induced by high concentrations of ACh. The significant difference between the half amplitude dose (K0.5) observed in the dose effect curves with ACh for Is inhibition (K0.5 = 8 X 10(-8) M) and for ACh-induced extra-current (K0.5 - 10(-6) M) may indicate the presence of two muscarinic receptors.

  12. [Regulation of acetylcholine synthesis in presynaptic endings of cholinergic neurons of the central nervous system].

    Science.gov (United States)

    Tuchek, S; Dolezhal, V; Richny, Ia

    1984-01-01

    Data on the acetylcholine (ACh) synthesis in nerve cells with special attention to its control are summarized in the paper. At rest or during moderate synaptic activity, the concentration of ACh in the compartment of its synthesis probably corresponds to the equilibrium between the substrates and products in the reaction catalysed by choline acetyltransferase. The release of ACh is followed by a transfer of ACh from the compartment of its synthesis to the compartment of release, and, automatically, by the synthesis of new ACh until a new equilibrium is reached in the compartment of synthesis. In addition, synaptic activity and the release of ACh support the synthesis of new ACh in the following ways: choline carriers are disinhibited by lowering the concentration of ACh in the nerve endings, and the transport of choline from the extracellular fluid to the cell interior according to its electro-chemical gradient is thus facilitated; the concentration of choline in the extracellular fluid is increased in the vicinity of the nerve endings as a consequence of the hydrolysis of the released ACh; postactivation hyperpolarization of the nerve endings brings about an increase of the choline transport and concentration in the nerve endings; presumably, the stimulation of muscarinic receptors brings about a further increase in the choline concentration in the vicinity of the nerve endings by the phosphatidylcholine hydrolysis intensification in postsynaptic cells; the decrease in the concentration of acetyl-CoA (as a consequence of the resynthesis of ACh) increases the activity of pyruvate dehydrogenase and the production of acetyl-CoA; conceivably, the increase in the concentration of Ca2+ ions in the nerve endings assists direct passage of acetyl-CoA from the mitochondria to the cytosol of the nerve endings, where the synthesis of ACh occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    Institute of Scientific and Technical Information of China (English)

    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.

  14. Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation.

    Science.gov (United States)

    Cheng, Kunrong; Samimi, Roxana; Xie, Guofeng; Shant, Jasleen; Drachenberg, Cinthia; Wade, Mark; Davis, Richard J; Nomikos, George; Raufman, Jean-Pierre

    2008-09-01

    Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

  15. Nicotine facilitates nicotinic acetylcholine receptor targeting to mitochondria but makes them less susceptible to selective ligands.

    Science.gov (United States)

    Uspenska, Kateryna; Lykhmus, Olena; Gergalova, Galyna; Chernyshov, Volodymyr; Arias, Hugo R; Komisarenko, Sergiy; Skok, Maryna

    2017-08-24

    Several nicotinic acetylcholine receptor (nAChR) subtypes are expressed in mitochondria to regulate the internal pathway of apoptosis in ion channel-independent manner. However, the mechanisms of nAChR activation in mitochondria and targeting to mitochondria are still unknown. Nicotine has been shown to favor nAChR pentamer assembly, folding, and maturation on the way of biosynthesis. The idea of the present work was to determine whether nicotine affects the content, glycosylation, and function of mitochondrial nAChRs. Experiments were performed in isolated liver mitochondria from mice, that either consumed or not nicotine with the drinking water (200μL/L) for 7days. Mitochondria detergent lysates were studied by sandwich or lectin ELISA for the presence and carbohydrate composition of different nAChR subunits. Intact mitochondria were examined by flow cytometry for the binding of fluorescently labeled α-cobratoxin and were tested in functional assay of cytochrome c release under the effect of either Ca 2+ or wortmannin in the presence or absence of nAChR-selective ligands, including PNU-282987 (1nM), dihydro-β-erythroidine (DhβE, 1μM), PNU-120596 (0.3, 3, or 10μM) and desformylflustrabromine hydrochloride (dFBr, 0.001, 0.3, or 1μM). It was found that nicotine consumption increased the ratio of mitochondrial vs non-mitochondrial nAChRs in the liver, enhanced fucosylation of mitochondrial nAChRs, but prevented the binding of α-cobratoxin and the cytochrome c release-attenuating effects of nAChR-specific agonists, antagonists, or positive allosteric modulators. It is concluded that nicotine consumption in vivo favors nAChR glycosylation and trafficking to mitochondria but makes them less susceptible to the effects of specific ligands. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Effect of donepezil hydrochloride (E2020) on extracellular acetylcholine concentration in the cerebral cortex of rats.

    Science.gov (United States)

    Kosasa, T; Kuriya, Y; Yamanishi, Y

    1999-10-01

    Donepezil hydrochloride (donepezil), a potent and selective acetylcholinesterase inhibitor, has been developed for the treatment of Alzheimer's disease. We studied the effect of oral administration of this drug on the extracellular acetylcholine (ACh) concentration in the cerebral cortex of rats using microdialysis. We also observed fasciculation, a peripheral cholinergic sign induced by activation of neuromuscular transmission, after oral administration of the drug as an index of peripheral cholinergic activation. Other cholinesterase inhibitors, tacrine, ENA-713 and TAK-147, were used as reference drugs. Donepezil significantly and dose-dependently increased the extracellular ACh concentration in the rat cerebral cortex within the dose range of 2.5-10 mg/kg. Tacrine, ENA-713 and TAK-147 also elevated the extracellular concentration of ACh. The minimum effective doses of donepezil, tacrine, ENA-713 and TAK-147 were (< or = 2.5, 10, 10 and < or = 10 mg/kg, respectively. Donepezil produced fasciculation at doses of 2.5 mg/kg and above, with a dose-dependent increase in incidence and intensity. The reference compounds also induced fasciculation in a dose-dependent manner. The threshold doses of tacrine, ENA-713 and TAK-147 for fasciculation were 5, 2.5 and 2.5 mg/kg, respectively. The values of the ratio of the minimum effective dose for the ACh-increasing action to that for the fasciculation-producing action were: donepezil, < or = 1; tacrine, 2; ENA-713, 4; TAK-147, < or = 4. These results indicate that orally administered donepezil has a potent and selective activity on the central cholinergic system.

  17. Nicotinic acetylcholine receptor availability in cigarette smokers: effect of heavy caffeine or marijuana use.

    Science.gov (United States)

    Brody, Arthur L; Hubert, Robert; Mamoun, Michael S; Enoki, Ryutaro; Garcia, Lizette Y; Abraham, Paul; Young, Paulina; Mandelkern, Mark A

    2016-09-01

    Upregulation of α4β2* nicotinic acetylcholine receptors (nAChRs) is one of the most well-established effects of chronic cigarette smoking on the brain. Prior research by our group gave a preliminary indication that cigarette smokers with concomitant use of caffeine or marijuana have altered nAChR availability. We sought to determine if smokers with heavy caffeine or marijuana use have different levels of α4β2* nAChRs than smokers without these drug usages. One hundred and one positron emission tomography (PET) scans, using the radiotracer 2-FA (a ligand for β2*-containing nAChRs), were obtained from four groups of males: non-smokers without heavy caffeine or marijuana use, smokers without heavy caffeine or marijuana use, smokers with heavy caffeine use (mean four coffee cups per day), and smokers with heavy marijuana use (mean 22 days of use per month). Total distribution volume (Vt/fp) was determined for the brainstem, prefrontal cortex, and thalamus, as a measure of nAChR availability. A significant between-group effect was found, resulting from the heavy caffeine and marijuana groups having the highest Vt/fp values (especially for the brainstem and prefrontal cortex), followed by smokers without such use, followed by non-smokers. Direct between-group comparisons revealed significant differences for Vt/fp values between the smoker groups with and without heavy caffeine or marijuana use. Smokers with heavy caffeine or marijuana use have higher α4β2* nAChR availability than smokers without these drug usages. These findings are likely due to increased nicotine exposure but could also be due to an interaction on a cellular/molecular level.

  18. Novel biomimetic composite material for potentiometric screening of acetylcholine, a neurotransmitter in Alzheimer's disease.

    Science.gov (United States)

    Sacramento, Ana S; Moreira, Felismina T C; Guerreiro, Joana L; Tavares, Ana P; Sales, M Goreti F

    2017-10-01

    This work describes a novel approach to produce an antibody-like biomimetic material. It includes preparing composite imprinted material never presented before, with highly conductive support nanostructures and assembling a high conductivity polymeric layer at low temperature. Overall, such highly conductive material may enhance the final features of electrically-based devices. Acetylcholine (ACh) was selected as target analyte, a neurotransmitter of importance in Alzheimer's disease. Potentiometric transduction was preferred, allowing quick responses and future adaptation to point-of-care requirements. The biomimetic material was obtained by bulk polymerization, where ACh was placed in a composite matrix of multiwalled carbon nanotubes (MWCNTs) and aniline (ANI). Subsequent polymerization, initiated by radical species, yielded a polymeric structure of polyaniline (PANI) acting as physical support of the composite. A non-imprinted material (NIM) having only PANI/MWCNT (without ACh) has been prepared for comparison of the biomimetic-imprinted material (BIM). RAMAN and Fourier Transform Infrared spectroscopy (FTIR), Transmission Electron microscopy (TEM), and Scanning Electron microscope (SEM) analysis characterized the structures of the materials. The ability of this biomaterial to rebind ACh was confirmed by including it as electroactive compound in a PVC/plasticizer mixture. The membranes with imprinted material and anionic additive presented the best analytical characteristics, with a sensitivity of 83.86mV decade -1 and limit of detection (LOD) of 3.45×10 -5 mol/L in HEPES buffer pH4.0. Good selectivity was observed against creatinine, creatine, glucose, cysteine and urea. The electrodes were also applied on synthetic serum samples and seemed a reliable tool for screening ACh in synthetic serum samples. The overall performance showed fast response, reusability, simplicity and low price. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Degradation rate of acetylcholine receptors inserted into denervated vertebrate neuromuscular junctions

    International Nuclear Information System (INIS)

    Shyng, S.L.; Salpeter, M.M.

    1989-01-01

    Many studies exist on the effect of denervation on the degradation of acetylcholine receptors (AChRs) at the vertebrate neuromuscular junction (nmj). These studies have described the behavior of either the total population of junctional receptors at different times after denervation, or of the receptors present at the time of denervation. No experimental studies yet exist on the degradation rate of the receptors newly inserted into denervated junctions. In the previous studies, the original receptors of mouse sternomastoid muscles were found to retain the slow degradation (t 1/2) of approximately 8-10 d of innervated junctional receptors for up to 10 d after denervation before accelerating to a t 1/2 of approximately 3 d. The total junctional receptors, on the other hand, showed a progressive increase in degradation rate from a t 1/2 of 8-10 d to a t 1/2 of 1 d. To reconcile these earlier observations, the present study examines the degradation of new receptors inserted into the nmj after denervation. To avoid possible contamination of the data with postdenervation extrajunctional receptors, we used transmission electron microscope autoradiography to study only receptors located at the postjunctional fold of the nmj. We established that the new receptors inserted into denervated junctions have a t 1/2 of approximately 1 d, considerably faster than that of the original receptors and equivalent to that of postdenervation extrajunctional receptors. Both original and new receptors are interspersed at the top of the junctional folds. Thus, until all the original receptors are degraded, the postjunctional membrane contains two populations of AChRs that maintain a total steady-state site density but degrade at different rates

  20. Blockade of central nicotine acetylcholine receptor signaling attenuate ghrelin-induced food intake in rodents.

    Science.gov (United States)

    Dickson, S L; Hrabovszky, E; Hansson, C; Jerlhag, E; Alvarez-Crespo, M; Skibicka, K P; Molnar, C S; Liposits, Z; Engel, J A; Egecioglu, E

    2010-12-29

    Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotine acetylcholine receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens, partly via cholinergic VTA afferents originating in the laterodorsal tegmental area (LDTg). Given that these cholinergic projections to the VTA have been implicated in natural as well as drug-induced reinforcement, we sought to investigate the role of cholinergic signaling in ghrelin-induced food intake as well as fasting-induced food intake, for which endogenous ghrelin has been implicated. We found that i.p. treatment with the non-selective centrally active nAChR antagonist, mecamylamine decreased fasting-induced food intake in both mice and rats. Moreover, central administration of mecamylamine decreased fasting-induced food intake in rats. I.c.v. ghrelin-induced food intake was suppressed by mecamylamine i.p. but not by hexamethonium i.p., a peripheral nAChR antagonist. Furthermore, mecamylamine i.p. blocked food intake following ghrelin injection into the VTA. Expression of the ghrelin receptor, the growth hormone secretagogue receptor 1A, was found to co-localize with choline acetyltransferase, a marker of cholinergic neurons, in the LDTg. Finally, mecamylamine treatment i.p. decreased the ability of palatable food to condition a place preference. These data suggest that ghrelin-induced food intake is partly mediated via nAChRs and that nicotinic blockade decreases the rewarding properties of food. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  1. The expression, localization and function of α7 nicotinic acetylcholine receptor in rat corpus cavernosum.

    Science.gov (United States)

    Faghir-Ghanesefat, Hedyeh; Rahimi, Nastaran; Yarmohammadi, Fatemeh; Mokhtari, Tahmineh; Abdollahi, Ali Reza; Ejtemaei Mehr, Shahram; Dehpour, Ahmad R

    2017-12-01

    Alpha7 nicotinic acetylcholine receptor (α7-nAChR), an emerging pharmacological target for a variety of medical conditions, is expressed in the most mammalian tissues with different effects. So, this study was designed to investigate the expression, localization and effect of α7-nAChR in rat corpus cavernosum (CC). Reverse transcription polymerase chain reaction (RT-PCR) revealed that α7-nAChR was expressed in rat CC and double immunofluorescence studies demonstrated the presence of α7-nAChR in corporal neurons. The rat CC segments were mounted in organ bath chambers and contracted with phenylephrine (0.1 μm -300 μm) to investigate the relaxation effect of electrical field stimulation (EFS,10 Hz) assessed in the presence of guanethidine (adrenergic blocker, 5 μm) and atropine (muscarinic cholinergic blocker, 1 μm) to obtain non-adrenergic non-cholinergic (NANC) response. Cumulative administration of nicotine significantly potentiated the EFS-induced NANC relaxation (-log EC50 = 7.5 ± 0.057). Whereas, the potentiated NANC relaxation of nicotine was significantly inhibited with different concentrations of methyllycaconitine citrate (α7-nAChR antagonist, P < 0.05) in preincubated strips. L-NAME (non-specific nitric oxide synthase inhibitor, 1 μm) completely blocked the neurogenic relaxation induced by EFS plus nicotine. To conclude α7-nAChR is expressed in rat CC and modulates the neurogenic relaxation response to nicotine. © 2017 Royal Pharmaceutical Society.

  2. Resistance to cycloxaprid in Laodelphax striatellus is associated with altered expression of nicotinic acetylcholine receptor subunits.

    Science.gov (United States)

    Zhang, Yueliang; Han, Yangchun; Yang, Qiong; Wang, Lihua; He, Peng; Liu, Zewen; Li, Zhong; Guo, Huifang; Fang, Jichao

    2018-04-01

    Cycloxaprid is a new oxabridged cis-configuration neonicotinoid insecticide, the resistance development potential and underlying resistance mechanism of which were investigated in the small brown planthopper, Laodelphax striatellus (Fallén), an important agricultural pest of rice. A cycloxaprid-resistant strain (YN-CPD) only achieved 10-fold higher resistance, in contrast to 106-fold higher resistance to buprofezin and 332-fold higher resistance to chlorpyrifos achieved after exposure to similar selection pressure, and the cycloxaprid selected line showed no cross-resistance to the buprofezin and chlorpyrifos-selected resistance strains. Moreover, we identified 10 nicotinic acetylcholine receptor (nAChR) subunits from the transcriptome of L. striatellus, and six segments had open reading frames (ORFs). While we did not find mutations in the nAChR genes of L. striatellus, subunits Lsα1 and Lsβ1 exhibited, respectively, 9.60-fold and 3.36-fold higher expression in the resistant strain, while Lsα8 exhibited 0.44-fold lower expression. Suppression of Lsα1 through ingestion of dsLsα1 led to an increase in susceptibility to cycloxaprid. The findings indicate that resistance to cycloxaprid develops slowly compared with resistance to other chemicals and without cross-resistance to chlorpyrifos or buprofezin; over-expressed Lsα1 is associated with low cycloxaprid resistance levels, but the importance of over-expressed Lsβ1 and reduced expression of Lsα8 could not be excluded. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  3. Selective expression of muscarinic acetylcholine receptor subtype M3 by mouse type III taste bud cells.

    Science.gov (United States)

    Mori, Yusuke; Eguchi, Kohgaku; Yoshii, Kiyonori; Ohtubo, Yoshitaka

    2016-11-01

    Each taste bud cell (TBC) type responds to a different taste. Previously, we showed that an unidentified cell type(s) functionally expresses a muscarinic acetylcholine (ACh) receptor subtype, M3, and we suggested the ACh-dependent modification of its taste responsiveness. In this study, we found that M3 is expressed by type III TBCs, which is the only cell type that possesses synaptic contacts with taste nerve fibers in taste buds. The application of ACh to the basolateral membrane of mouse fungiform TBCs in situ increased the intracellular Ca 2+ concentration in 2.4 ± 1.4 cells per taste bud (mean ± SD, n = 14). After Ca 2+ imaging, we supravitally labeled type II cells (phospholipase C β2 [PLCβ2]-immunoreactive cells) with Lucifer yellow CH (LY), a fluorescent dye and investigated the positional relationship between ACh-responding cells and LY-labeled cells. After fixation, the TBCs were immunohistostained to investigate the positional relationships between immunohistochemically classified cells and LY-labeled cells. The overlay of the two positional relationships obtained by superimposing the LY-labeled cells showed that all of the ACh-responding cells were type III cells (synaptosomal-associated protein 25 [SNAP-25]-immunoreactive cells). The ACh responses required no added Ca 2+ in the bathing solution. The addition of 1 μM U73122, a phospholipase C inhibitor, decreased the magnitude of the ACh response, whereas that of 1 μM U73343, a negative control, had no effect. These results suggest that type III cells respond to ACh and release Ca 2+ from intracellular stores. We also discuss the underlying mechanism of the Ca 2+ response and the role of M3 in type III cells.

  4. Elemental maps in human allantochorial placental vessels cells: 1. High K{sup +} and acetylcholine effects

    Energy Technology Data Exchange (ETDEWEB)

    Michelet-Habchi, C. E-mail: michelet@cenbg.in2p3.fr; Barberet, Ph.; Dutta, R.K.; Guiet-Bara, A.; Bara, M.; Moretto, Ph

    2003-09-01

    Regulation of vascular tone in the fetal extracorporeal circulation most likely depends on circulating hormones, local paracrine mechanisms and changes in membrane potential of vascular smooth muscle cells (VSMCs) and of vascular endothelial cells (VECs). The membrane potential is a function of the physiological activities of ionic channels (particularly, K{sup +} and Ca{sup 2+} channels in these cells). These channels regulate the ionic distribution into these cells. Micro-particle induced X-ray emission (PIXE) analysis was applied to determine the ionic composition of VSMC and of VEC in the placental human allantochorial vessels in a physiological survival medium (Hanks' solution) modified by the addition of acetylcholine (ACh: which opens the calcium-sensitive K{sup +} channels, K{sub Ca}) and of high concentration of K{sup +} (which blocks the voltage-sensitive K{sup +} channels, K{sub df}). In VSMC (media layer), the addition of ACh induced no modification of the Na, K, Cl, P, S, Mg and Ca concentrations and high K{sup +} medium increased significantly the Cl and K concentrations, the other ion concentrations remaining constant. In endothelium (VEC), ACh addition implicated a significant increase of Na and K concentration, and high K{sup +} medium, a significant increase in Cl and K concentration. These results indicated the importance of K{sub df}, K{sub Ca} and K{sub ATP} channels in the regulation of K{sup +} intracellular distribution in VSMC and VEC and the possible intervention of a Na-K-2Cl cotransport and corroborated the previous electrophysiological data.

  5. Pharmacological Characterisation of Nicotinic Acetylcholine Receptors Expressed in Human iPSC-Derived Neurons.

    Directory of Open Access Journals (Sweden)

    Anna Chatzidaki

    Full Text Available Neurons derived from human induced pluripotent stem cells (iPSCs represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels. Previous studies on iPSC-derived neuronal cells have reported the functional characterisation of a variety of receptors and ion channels, including glutamate receptors, γ-aminobutyric acid (GABA receptors and several voltage-gated ion channels. In the present study we have examined the expression and functional properties of nicotinic acetylcholine receptors (nAChRs in human iPSC-derived neurons. Gene expression analysis indicated the presence of transcripts encoding several nAChR subunits, with highest levels detected for α3-α7, β1, β2 and β4 subunits (encoded by CHRNA3-CHRNA7, CHRNB1, CHRNB2 and CHRNB4 genes. In addition, similarly high transcript levels were detected for the truncated dupα7 subunit transcript, encoded by the partially duplicated gene CHRFAM7A, which has been associated with psychiatric disorders such as schizophrenia. The functional properties of these nAChRs have been examined by calcium fluorescence and by patch-clamp recordings. The data obtained suggest that the majority of functional nAChRs expressed in these cells have pharmacological properties typical of α7 receptors. Large responses were induced by a selective α7 agonist (compound B, in the presence of the α7-selective positive allosteric modulator (PAM PNU-120596, which were blocked by the α7-selective antagonist methyllycaconitine (MLA. In addition, a small proportion of the neurons express nAChRs with properties typical of heteromeric (non-α7 containing nAChR subtypes. These cells therefore represent a great tool to advance our understanding of the properties of native human nAChRs, α7 in particular.

  6. Orthosteric and Allosteric Ligands of Nicotinic Acetylcholine Receptors for Smoking Cessation

    Directory of Open Access Journals (Sweden)

    Tasnim S. Mohamed

    2015-11-01

    Full Text Available Nicotine addiction, the result of tobacco use, leads to over six million premature deaths world-wide, a number that is expected to increase by a third within the next two decades. While more than half of smokers want and attempt to quit, only a small percentage of smokers are able to quit without pharmacological interventions. Therefore, over the past decades, researchers in academia and the pharmaceutical industry have focused their attention on the development of more effective smoking cessation therapies, which is now a growing 1.9 billion dollar market. Because the role of neuronal nicotinic acetylcholine receptors (nAChR in nicotine addiction is well established, nAChR based therapeutics remain the leading strategy for smoking cessation. However, the development of neuronal nAChR drugs that are selective for a nAChR subpopulation is challenging, and only few neuronal nAChR drugs are clinically available. Among the many neuronal nAChR subtypes that have been identified in the brain, the α4β2 subtype is the most abundant and plays a critical role in nicotine addiction. Here, we review the role of neuronal nAChRs, especially the α4β2 subtype, in the development and treatment of nicotine addiction. We also compare available smoking cessation medications and other nAChR orthosteric and allosteric ligands that have been developed with emphasis on the difficulties faced in the development of clinically useful compounds with high nAChR subtype selectivity.

  7. Cognitive effects of dopamine depletion in the context of diminished acetylcholine signaling capacity in mice

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    Lilia Zurkovsky

    2013-01-01

    A subset of patients with Parkinson’s disease acquires a debilitating dementia characterized by severe cognitive impairments (i.e. Parkinson’s disease dementia; PDD. Brains from PDD patients show extensive cholinergic loss as well as dopamine (DA depletion. We used a mutant mouse model to directly test whether combined cholinergic and DA depletion leads to a cognitive profile resembling PDD. Mice carrying heterozygous deletion of the high-affinity, hemicholinium-3-sensitive choline transporter (CHTHET show reduced levels of acetylcholine throughout the brain. We achieved bilateral DA depletion in CHTHET and wild-type (WT littermates via intra-striatal infusion of 6-hydroxydopamine (6-OHDA, or used vehicle as control. Executive function and memory were evaluated using rodent versions of cognitive tasks commonly used with human subjects: the set-shifting task and spatial and novel-object recognition paradigms. Our studies revealed impaired acquisition of attentional set in the set-shifting paradigm in WT-6OHDA and CHTHET-vehicle mice that was exacerbated in the CHTHET-6OHDA mice. The object recognition test following a 24-hour delay was also impaired in CHTHET-6OHDA mice compared with all other groups. Treatment with acetylcholinesterase (AChE inhibitors physostigmine (0.05 or 0.1 mg/kg and donepezil (0.1 and 0.3 mg/kg reversed the impaired object recognition of the CHTHET-6OHDA mice. Our data demonstrate an exacerbated cognitive phenotype with dual ACh and DA depletion as compared with either insult alone, with traits analogous to those observed in PDD patients. The results suggest that combined loss of DA and ACh could be sufficient for pathogenesis of specific cognitive deficits in PDD.

  8. The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters

    Directory of Open Access Journals (Sweden)

    Madhavan Raghavan

    2008-07-01

    Full Text Available Abstract Background A crucial event in the development of the vertebrate neuromuscular junction (NMJ is the postsynaptic enrichment of muscle acetylcholine (ACh receptors (AChRs. This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown. Results Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877 blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1 also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly. Conclusion Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.

  9. Effects of Acetylcholine and Noradrenalin on Action Potentials of Isolated Rabbit Sinoatrial and Atrial Myocytes

    Science.gov (United States)

    Verkerk, Arie O.; Geuzebroek, Guillaume S. C.; Veldkamp, Marieke W.; Wilders, Ronald

    2012-01-01

    The autonomic nervous system controls heart rate and contractility through sympathetic and parasympathetic inputs to the cardiac tissue, with acetylcholine (ACh) and noradrenalin (NA) as the chemical transmitters. In recent years, it has become clear that specific Regulators of G protein Signaling proteins (RGS proteins) suppress muscarinic sensitivity and parasympathetic tone, identifying RGS proteins as intriguing potential therapeutic targets. In the present study, we have identified the effects of 1 μM ACh and 1 μM NA on the intrinsic action potentials of sinoatrial (SA) nodal and atrial myocytes. Single cells were enzymatically isolated from the SA node or from the left atrium of rabbit hearts. Action potentials were recorded using the amphotericin-perforated patch-clamp technique in the absence and presence of ACh, NA, or a combination of both. In SA nodal myocytes, ACh increased cycle length and decreased diastolic depolarization rate, whereas NA decreased cycle length and increased diastolic depolarization rate. Both ACh and NA increased maximum upstroke velocity. Furthermore, ACh hyperpolarized the maximum diastolic potential. In atrial myocytes stimulated at 2 Hz, both ACh and NA hyperpolarized the maximum diastolic potential, increased the action potential amplitude, and increased the maximum upstroke velocity. Action potential duration at 50 and 90% repolarization was decreased by ACh, but increased by NA. The effects of both ACh and NA on action potential duration showed a dose dependence in the range of 1–1000 nM, while a clear-cut frequency dependence in the range of 1–4 Hz was absent. Intermediate results were obtained in the combined presence of ACh and NA in both SA nodal and atrial myocytes. Our data uncover the extent to which SA nodal and atrial action potentials are intrinsically dependent on ACh, NA, or a combination of both and may thus guide further experiments with RGS proteins. PMID:22754533

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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)

  12. Detection of Ca2+-induced acetylcholine released from leukemic T-cells using an amperometric microfluidic sensor.

    Science.gov (United States)

    Akhtar, Mahmood H; Hussain, Khalil K; Gurudatt, N G; Shim, Yoon-Bo

    2017-12-15

    A microfluidic structured-dual electrodes sensor comprising of a pair of screen printed carbon electrodes was fabricated to detect acetylcholine, where one of them was used for an enzyme reaction and another for a detection electrode. The former was coated with gold nanoparticles and the latter with a porous gold layer, followed by electropolymerization of 2, 2:5,2-terthiophene-3-(p-benzoic acid) (pTTBA) on both the electrodes. Then, acetylcholinesterase was covalently attached onto the reaction electrode, and hydrazine and choline oxidase were co-immobilized on the detection electrode. The layers of both modified electrodes were characterized employing voltammetry, field emission scanning electron microscopy, X-ray photoelectron spectroscopy, and quartz crystal microscopy. After the modifications of both electrode surfaces, they were precisely faced each other to form a microfluidic channel structure, where H 2 O 2 produced from the sequential enzymatic reactions was reduced by hydrazine to obtain the analytical signal which was analyzed by the detection electrode. The microfluidic sensor at the optimized experimental conditions exhibited a wide dynamic range from 0.7nM to 1500μM with the detection limit of 0.6 ± 0.1nM based on 3s (S/N = 3). The biomedical application of the proposed sensor was evaluated by detecting acetylcholine in human plasma samples. Moreover, the Ca 2+ -induced acetylcholine released in leukemic T-cells was also investigated to show the in vitro detection ability of the designed microfluidic sensor. Interference due to the real component matrix were also studied and long term stability of the designed sensor was evaluated. The analytical performance of the designed sensor was also compared with commercially available ACh detection kit. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Prefrontal cortex, caloric restriction and stress during aging: studies on dopamine and acetylcholine release, BDNF and working memory.

    Science.gov (United States)

    Del Arco, Alberto; Segovia, Gregorio; de Blas, Marta; Garrido, Pedro; Acuña-Castroviejo, Dario; Pamplona, Reinald; Mora, Francisco

    2011-01-01

    This study was designed to investigate whether long-term caloric restriction during the life span of the rat changes the effects of an acute mild stress on the release of dopamine and acetylcholine in the prefrontal cortex (PFC) and on working memory performance. Spontaneous motor activity was also monitored and levels of BDNF measured in the prefrontal cortex, amygdala and hippocampus. Male Wistar rats (3 months of age) were housed during 3, 12, 21 and 27 months (6, 15, 24 and 30 months of age at the end of housing) in caloric restriction (CR; 40% food intake restriction) or control conditions. After behavioural testing, animals were further subdivided into two other groups. In one of the groups BDNF protein levels were determined. In the other group rats were implanted with guide cannulas into the PFC to perform microdialysis experiments. In CR rats the release of dopamine produced by handling stress did not differ from the response found in control rats of 6, 15 and 24 months of age. The release of acetylcholine was not changed at the ages of 6 and 15 months but reduced at the age of 24 months. Stress did not change dopamine or acetylcholine release in CR and control rats of 30 months of age. BDNF levels were increased in the hippocampus and amygdala, but not in the PFC, of 6 and 15 months CR rats. Spontaneous motor activity was increased in all groups of CR rats. Age, however, decreased motor activity in CR and control rats. Both experimental groups showed similar working memory performance in a delayed alternation task in basal conditions and after a situation of acute stress. These results suggest that CR does not modify the function of the PFC in response to an acute stress nor the changes found as a result of the normal process of aging. Copyright © 2010 Elsevier B.V. All rights reserved.

  14. Selective breeding of two lines of guinea pigs differing in bronchial sensitivity to acetylcholine and histamine exposure.

    Science.gov (United States)

    Mikami, H; Nishibata, R; Kawamoto, Y; Ino, T

    1991-04-01

    We developed two lines of guinea pigs, one as model animals for bronchial asthma with bronchial hypersensitivity and the other with hyposensitivity as a control. In the last four years, the bronchial hypersensitive line (BHS) and hyposensitive line (BHR), both derived from Hartley strain guinea pigs, have been selected by using bronchial reactivity to acetylcholine and to histamine as parameters. Both lines have reached the F6 generation. The following results were obtained with the two lines: 1) Sib and cous in matings, and mating of selected consanguineous individuals were adopted in breeding BHS and BHR. The breeding started with six families, each, but in the F6 generation the number of families decreased to two in each line. 2) Appearance rates of hyper- or hyposensitivity to acetylcholine and histamine increased with successive generations in both lines, which had been completely separated by the F6 generation. 3) Coefficients of inbreeding in BHS and BHR in the F6 generation ranged from 42% to 45% in the former and 42% in the latter. 4) Heritabilities (h2) of BHS and BHR for the appearance rates of sensitivity to acetylcholine were presumed to be 0.54 in the former and 0.69 in the latter. 5) No difference in the body weight of 0, 20, and 40 day-old BHS was observed in any generation. On the other hand, the body weight of 20 and 40 day-old BHR tended to decrease with successive generations. 6) Mean litter sizes of BHS and BHR in each of the generations ranged from 2.24 to 3.47 animals in the former and from 2.63 to 3.38 animals in the latter.(ABSTRACT TRUNCATED AT 250 WORDS)

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

    International Nuclear Information System (INIS)

    Bataller, Georges

    1971-10-01

    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) [fr

  16. Modulative effects of acetylcholine and GABA upon homeostatic patterns in nerve tissue under the conditions of low-intensive irradiation

    International Nuclear Information System (INIS)

    Dvorets'kij, A.Yi.; Anan'jeva, T.V.

    1999-01-01

    Investigation of both the acetylcholine and γ-aminobutyric acid modulative effects upon the K + content and Na, K-pump activity in rat brain cortex slices after single prolonged X-irradiation or chronic (fractionated) one with doses of 25 cGy and 50 cGy (1.75 mGy/min) resulted in considerable modification of the metabolic regulation processes. A role of synaptic neuro mediators in supporting the nerve cell functional activity via modulation of the anti gradient K + transport and intracellular potassium homeostasis under any harmful factor influence is discussed

  17. Ablation of the Right Cardiac Vagus Nerve Reduces Acetylcholine Content without Changing the Inflammatory Response during Endotoxemia

    Directory of Open Access Journals (Sweden)

    Konstanze Plaschke

    2018-02-01

    Full Text Available Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a “non-neuronal cardiac cholinergic system” that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg body weight, intravenously and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory (IL-1β, IL-6, TNF-α cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX led to a marked decrease in heart

  18. Ablation of the Right Cardiac Vagus Nerve Reduces Acetylcholine Content without Changing the Inflammatory Response during Endotoxemia.

    Science.gov (United States)

    Plaschke, Konstanze; Do, Thuc Quyen Monica; Uhle, Florian; Brenner, Thorsten; Weigand, Markus A; Kopitz, Jürgen

    2018-02-01

    Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a "non-neuronal cardiac cholinergic system" that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX) with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg body weight, intravenously) and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory (IL-1β, IL-6, TNF-α) cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX) led to a marked decrease in heart acetylcholine

  19. Investigation of the presence and antinociceptive function of muscarinic acetylcholine receptors in the African naked mole-rat (Heterocephalus glaber)

    DEFF Research Database (Denmark)

    Jørgensen, Kristine B.; Krogh-Jensen, Karen; Pickering, Darryl S

    2016-01-01

    The present study investigated the cholinergic system in the African naked mole-rat (Heterocephalus glaber) with focus on the muscarinic acetylcholine receptor subtypes M1 and M4. The protein sequences for the subtypes m 1–5 of the naked mole-rat were compared to that of the house mouse (Mus...... musculus) using basic local alignment search tool (BLAST). The presence and function of M1 and M4 was investigated in vivo, using the formalin test with the muscarinic receptor agonists xanomeline and VU0152100. Spinal cord tissue from the naked mole-rat was used for receptor saturation binding studies...

  20. The main immunogenic region of acetylcholine receptors does not provoke the formation of antibodies of a predominant idiotype.

    Science.gov (United States)

    Killen, J A; Hochschwender, S M; Lindstrom, J M

    1985-08-01

    Anti-idiotype antibodies were induced in rats by immunization with rat monoclonal antibodies to the main immunogenic region of acetylcholine receptors. These anti-idiotype antibodies showed very little crossreaction with other rat monoclonal antibodies which bind to the same region of the receptor. When the rats producing these anti-idiotype antibodies were immunized with receptor, they showed no net decrease in anti-receptor antibody production. These data indicate that, although more than half of the antibodies produced by rats immunized with receptor are directed at a small region, many anti-receptor idiotypes are involved in this response and anti-idiotype therapy is not beneficial.

  1. The effect of acetylcholine, LatA and FAA on phloem assimilates translocation of Raphanus sativus L

    International Nuclear Information System (INIS)

    Yang Chongjun; Zhang Ping

    2005-01-01

    The petiole phloem of Raphanus sativus L. is treated with the medicaments of acetylcholine (Ach, the expansionist material of protoplasm), latrunculin A (LatA, the dissolubility of microfilament) and FAA (the regularization of phloem). The effects of treatments are measured by the accumulated content of dissoluble sugar and starch in the leaves, and 14 C-labelled assimilates. The regulating role of three medicaments on the translocation of assimilates in the phloem of Raphanus sativus L are investigated. The results indicate that low Ach improves assimilates translocation while LatA and FAA inhibit it in petiole phloem of Raphanus sativus L.. (authors)

  2. Myasthenic Crisis Complicated with Myxedema, Positive for Both Anti-acetylcholine Receptor and Anti-muscle-specific Tyrosine Kinase Antibodies.

    Science.gov (United States)

    Horiuchi, Kazuhiro; Nagai, Azusa; Wakita, Masahiro; Ito, Shotaro; Takamura, Kei; Houzen, Hideki

    2018-01-15

    We herein report the case of myasthenic crisis occurring in a 51-year-old man. He had experienced ptosis, increased body weight with edema, and fatigue with dyspnea. He presented at our emergency department with disturbed consciousness. He was originally diagnosed with myxedema coma, and he required artificial respiration. Because his weakness persisted and he was positive for anti-acetylcholine receptor antibodies and anti-muscle-specific tyrosine kinase antibodies, we diagnosed myasthenic crisis after various examinations. His clinical response to treatment was good and he was discharged in an ambulatory status 3 months after admission. This case demonstrates that myasthenic crisis may occur in association with myxedema.

  3. Effect of α-bungarotoxin and etorphine on acetylcholine-evoked release of endogenous and radiolabeled catecholamines from primary culture of adrenal chromaffin cells

    International Nuclear Information System (INIS)

    Kageyama, H.; Guidotti, A.

    1984-01-01

    Cell cultures of adrenal medulla have become an important research tool to study basic processes that regulate catecholamine storage, release and synthesis. Release has been studied either by labeling with [ 3 H]norepinephrine and measuring release of radioactivity or by measuring the endogenous catecholamines released with HPLC. Acetylcholine (5X10 -6 -10 -4 M) appears to release preferentially norepinephrine, although the cells store more epinephrine than norepinephrine. Etorphine and α-bungarotoxin antagonize the release of catecholamines elicited by acetylcholine. This inhibitory action appears to be greater when the measurement of endogenous catecholamines rather than radioactive norepinephrine is used to monitor the action of acetylcholine. The data suggest that the measurement of endogenous catecholamines by HPLC is preferable to the [ 3 H]NE loading and release technique, especially when analyzing the effects of low concentrations of drugs that are thought to affect nicotinic receptor function. (Auth.)

  4. Evidence that morphine and opioid peptides do not share a common pathway with adenosine in inhibiting acetylcholine release from isolated intestine.

    Science.gov (United States)

    Vizi, E S; Somogyi, G T; Magyar, K

    1981-12-01

    1 The release of acetylcholine from guinea-pig ileal isolated longitudinal muscle strip with intact Auerbach's plexus was measured by bioassay and by a radioisotope technique. 2 Normorphine (5 x 10(-7)M) and D-Met2, Pro5-enkephalinamide (D-Met, Pro-EA) reduced the release of acetylcholine. Theophylline, an adenosine antagonist, failed to prevent the inhibitory effect of normorphine or D-Met, Pro-EA. 3 Theophylline (1.7 x 10(-4)M) by itself enhanced the twitch responses to field stimulation (0.1 Hz) but did not prevent the inhibitory effect of normorphine and D-Met, Pro-EA. 4 From the results it can be concluded that morphine and opioid peptides do not share a common pathway with adenosine in inhibiting acetylcholine release from axon terminals of Auerbach's plexus.

  5. PI3K/Akt-independent NOS/HO activation accounts for the facilitatory effect of nicotine on acetylcholine renal vasodilations: modulation by ovarian hormones.

    Directory of Open Access Journals (Sweden)

    Eman Y Gohar

    Full Text Available We investigated the effect of chronic nicotine on cholinergically-mediated renal vasodilations in female rats and its modulation by the nitric oxide synthase (NOS/heme oxygenase (HO pathways. Dose-vasodilatory response curves of acetylcholine (0.01-2.43 nmol were established in isolated phenylephrine-preconstricted perfused kidneys obtained from rats treated with or without nicotine (0.5-4.0 mg/kg/day, 2 weeks. Acetylcholine vasodilations were potentiated by low nicotine doses (0.5 and 1 mg/kg/day in contrast to no effect for higher doses (2 and 4 mg/kg/day. The facilitatory effect of nicotine was acetylcholine specific because it was not observed with other vasodilators such as 5'-N-ethylcarboxamidoadenosine (NECA, adenosine receptor agonist or papaverine. Increases in NOS and HO-1 activities appear to mediate the nicotine-evoked enhancement of acetylcholine vasodilation because the latter was compromised after pharmacologic inhibition of NOS (L-NAME or HO-1 (zinc protoporphyrin, ZnPP. The renal protein expression of phosphorylated Akt was not affected by nicotine. We also show that the presence of the two ovarian hormones is necessary for the nicotine augmentation of acetylcholine vasodilations to manifest because nicotine facilitation was lost in kidneys of ovariectomized (OVX and restored after combined, but not individual, supplementation with medroxyprogesterone acetate (MPA and estrogen (E2. Together, the data suggests that chronic nicotine potentiates acetylcholine renal vasodilation in female rats via, at least partly, Akt-independent HO-1 upregulation. The facilitatory effect of nicotine is dose dependent and requires the presence of the two ovarian hormones.

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

    DEFF Research Database (Denmark)

    Ettrup, Anders; Mikkelsen, Jens D; Lehel, Szabolcs

    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......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)n...

  7. Dianicline, a novel α4β2 nicotinic acetylcholine receptor partial agonist, for smoking cessation: a randomized placebo-controlled clinical trial

    DEFF Research Database (Denmark)

    Tonstad, Serena; Holme, Ingar; Tønnesen, Philip

    2011-01-01

    Dianicline is a α4β2 nicotinic acetylcholine receptor partial agonist, a class of drugs that includes varenicline and cytisine. Varenicline is efficacious for smoking cessation, while cytisine has not been studied systematically. The efficacy of dianicline has not been previously tested in an ade......Dianicline is a α4β2 nicotinic acetylcholine receptor partial agonist, a class of drugs that includes varenicline and cytisine. Varenicline is efficacious for smoking cessation, while cytisine has not been studied systematically. The efficacy of dianicline has not been previously tested...

  8. 11C-NS14492 as a novel PET radioligand for imaging cerebral alpha7 nicotinic acetylcholine receptors: in vivo evaluation and drug occupancy measurements

    DEFF Research Database (Denmark)

    Ettrup, Anders; Mikkelsen, Jens D; Lehel, Szabolcs

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    International Nuclear Information System (INIS)

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-01

    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

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

    International Nuclear Information System (INIS)

    Ogawa, Mikako; Nishiyama, Shingo; Tsukada, Hideo; Hatano, Kentaro; Fuchigami, Takeshi; Yamaguchi, Hiroshi; Matsushima, Yoshitaka; Ito, Kengo; Magata, Yasuhiro

    2010-01-01

    Introduction: The nicotinic acetylcholine receptor (nAChR) α7 subtype (α 7 nAChR) is one of the major nAChR subtypes in the brain. We synthesized C-11 labeled α 7 nAChR ligands, (R)-2-[ 11 C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([ 11 C](R)-MeQAA) and its isomer (S)-[ 11 C]MeQAA, for in vivo investigation with positron emission tomography (PET). Then, the potential of (R)- and (S)-[ 11 C]MeQAA for in vivo imaging of α 7 nAChR in the brain was evaluated in mice and monkeys. Methods: The binding affinity for α 7 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 α 7 nAChR was 41 and 182 nM for (R)- and (S)-MeQAA, respectively. The initial uptake in the mouse brain was high ([ 11 C](R)-MeQAA: 7.68 and [ 11 C](S)-MeQAA: 6.65 %dose/g at 5 min). The clearance of [ 11 C](R)-MeQAA was slow in the hippocampus (α 7 nAChR-rich region) but was rapid in the cerebellum (α 7 nAChR-poor region). On the other hand, the clearance was fast for [ 11 C](S)-MeQAA in all regions. The brain uptake of [ 11 C](R)-MeQAA was decreased by methyllycaconitine (α 7 nAChR antagonist) treatment. In monkeys, α 7 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 [ 11 C](R)-MeQAA, while the uptake was rather homogeneous for [ 11 C](S)-MeQAA. Conclusions: [ 11 C](R)-MeQAA was successfully synthesized and showed high uptake to the brain. However, since the in vivo selectivity for α 7 nAChR was not enough, further PET kinetic analysis or structure optimization is needed for specific visualization of brain α 7 nAChRs in vivo.

  12. The α7-nicotinic acetylcholine receptor mediates the sensitivity of gastric cancer cells to taxanes.

    Science.gov (United States)

    Tu, Chao-Chiang; Huang, Chien-Yu; Cheng, Wan-Li; Hung, Chin-Sheng; Uyanga, Batzorig; Wei, Po-Li; Chang, Yu-Jia

    2016-04-01

    Gastric cancer is difficult to cure because most patients are diagnosed at an advanced disease stage. Systemic chemotherapy remains an important therapy for gastric cancer, but both progression-free survival and disease-free survival associated with various combination regimens are limited because of refractoriness and chemoresistance. Accumulating evidence has revealed that the homomeric α7-nicotinic acetylcholine receptor (A7-nAChR) promotes human gastric cancer by driving cancer cell proliferation, migration, and metastasis. Therefore, A7-nAChR may serve as a potential therapeutic target for gastric cancer. However, the role of A7-nAChR in taxane therapy for gastric cancer was unclear. Cells were subjected to A7-nAChR knockdown (A7-nAChR KD) using short interfering RNA (siRNA). The anti-proliferative effects of taxane were assessed via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL), and cell cycle distribution assays. A7-nAChR-KD cells exhibited low resistance to docetaxel and paclitaxel treatment, as measured by the MTT assay. Following paclitaxel treatment, the proportion of apoptotic cells was higher among A7-nAChR-KD cells than among scrambled control cells, as measured by cell cycle distribution and TUNEL assays. Further molecular analyses showed a reduction in the pAKT levels and a dramatic increase in the Bad levels in paclitaxel-treated A7-nAChR-KD cells but not in scrambled control cells. Following paclitaxel treatment, the level of Bax was slightly increased in both cell populations, whereas Poly (ADP-ribose) polymerase (PARP) cleavage was increased only in A7-nAChR-KD cells. These findings indicate that A7-nAChR-KD cells are more sensitive to paclitaxel treatment. We conclude that A7-nAChR may be a key biomarker for assessing the chemosensitivity of gastric cancer cells to taxane.

  13. (E)-[125I]-5-AOIBV: a SPECT radioligand for the vesicular acetylcholine transporter

    International Nuclear Information System (INIS)

    Emond, Patrick; Mavel, Sylvie; Zea-Ponce, Yolanda; Kassiou, Michael; Garreau, Lucette; Bodard, Sylvie; Drossard, Marie-Laure; Chalon, Sylvie; Guilloteau, Denis

    2007-01-01

    The premise that, over the course of Alzheimer's disease (AD), changes in the levels of the vesicular acetylcholine transporter (VAChT) occur in parallel with changes to other cholinergic marker proteins provides the basis for the applicability of benzovesamicol derivatives as radioligands for AD studies by single photon emission computed tomography or positron emission tomography. We report the synthesis of enantiopure benzovesamicol derivatives: (R,R) or (S,S)-(E)-2-hydroxy-5-(3-iodoprop-2-en-1-oxy)-3- (4-phenylpiperidino)tetralin [(R,R)-AOIBV: K d =0.45 nM or (S,S)-5-AOIBV: K d =4.3 nM] and their corresponding tributyltin precursors for radioiodination. (R,R or S,S)-5-AOIBV was labeled with iodine-125 from their corresponding n-tributyltin precursors. Both compounds were obtained with radiochemical and optical purity greater than 97% and in radiochemical yields ranging 34-36%. To determine if these compounds could provide an advantage when compared to [ 125 I]-iodo benzovesamicol (IBVM), IBVM was also labeled and used as the reference compound in all ex vivo experiments. Ex vivo biodistribution experiments in rats revealed that [ 125 I]-(R,R)-5-AOIBV displayed the most suitable pharmacological profile as the radioactivity distribution corresponded well with the known VAChT brain density. Moreover, pre-injection of vesamicol prevented the uptake of [ 125 I]-(R,R)-5-AOIBV in striatum, cortex and hippocampus, demonstrating selectivity for the VAChT. However, even if time activity curves of [ 125 I]-(R,R)-5-AOIBV confirmed that this compound could be used to visualize the VAChT in vivo, at each point of the kinetic study, [ 125 I]-(R,R)-5-AOIBV showed a lower specific binding compared to [ 125 I]-IBVM. These results made [ 125 I]-( R,R)-5-AOIBV inferior to [ 125 I]-IBVM for the VAChT exploration in vivo

  14. (E)-[{sup 125}I]-5-AOIBV: a SPECT radioligand for the vesicular acetylcholine transporter

    Energy Technology Data Exchange (ETDEWEB)

    Emond, Patrick [INSERM U619, 37000 Tours (France); Universite Francois-Rabelais de Tours, CHRU, Hopital Bretonneau, Service de Medecine nucleaire, 37000 Tours (France); Mavel, Sylvie [INSERM U619, 37000 Tours (France); Universite Francois-Rabelais de Tours, CHRU, Hopital Bretonneau, Service de Medecine nucleaire, 37000 Tours (France)], E-mail: sylvie.mavel@univ-tours.fr; Zea-Ponce, Yolanda [INSERM U619, 37000 Tours (France); Universite Francois-Rabelais de Tours, CHRU, Hopital Bretonneau, Service de Medecine nucleaire, 37000 Tours (France); Kassiou, Michael [Discipline of Medical Radiation Sciences, Brain and Mind Research Institute, University of Sydney, NSW 2050 (Australia); School of Chemistry, University of Sydney, NSW 2006 (Australia); Garreau, Lucette; Bodard, Sylvie; Drossard, Marie-Laure; Chalon, Sylvie; Guilloteau, Denis [INSERM U619, 37000 Tours (France); Universite Francois-Rabelais de Tours, CHRU, Hopital Bretonneau, Service de Medecine nucleaire, 37000 Tours (France)

    2007-11-15

    The premise that, over the course of Alzheimer's disease (AD), changes in the levels of the vesicular acetylcholine transporter (VAChT) occur in parallel with changes to other cholinergic marker proteins provides the basis for the applicability of benzovesamicol derivatives as radioligands for AD studies by single photon emission computed tomography or positron emission tomography. We report the synthesis of enantiopure benzovesamicol derivatives: (R,R) or (S,S)-(E)-2-hydroxy-5-(3-iodoprop-2-en-1-oxy)-3- (4-phenylpiperidino)tetralin [(R,R)-AOIBV: K{sub d}=0.45 nM or (S,S)-5-AOIBV: K{sub d}=4.3 nM] and their corresponding tributyltin precursors for radioiodination. (R,R or S,S)-5-AOIBV was labeled with iodine-125 from their corresponding n-tributyltin precursors. Both compounds were obtained with radiochemical and optical purity greater than 97% and in radiochemical yields ranging 34-36%. To determine if these compounds could provide an advantage when compared to [{sup 125}I]-iodo benzovesamicol (IBVM), IBVM was also labeled and used as the reference compound in all ex vivo experiments. Ex vivo biodistribution experiments in rats revealed that [{sup 125}I]-(R,R)-5-AOIBV displayed the most suitable pharmacological profile as the radioactivity distribution corresponded well with the known VAChT brain density. Moreover, pre-injection of vesamicol prevented the uptake of [{sup 125}I]-(R,R)-5-AOIBV in striatum, cortex and hippocampus, demonstrating selectivity for the VAChT. However, even if time activity curves of [{sup 125}I]-(R,R)-5-AOIBV confirmed that this compound could be used to visualize the VAChT in vivo, at each point of the kinetic study, [{sup 125}I]-(R,R)-5-AOIBV showed a lower specific binding compared to [{sup 125}I]-IBVM. These results made [{sup 125}I]-( R,R)-5-AOIBV inferior to [{sup 125}I]-IBVM for the VAChT exploration in vivo.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Taillebois, Emiliane; Beloula, Abdelhamid; Quinchard, Sophie; Jaubert-Possamai, Stéphanie; Daguin, Antoine; Servent, Denis; Tagu, Denis

    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 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. PMID:24801634

  17. Honeybees Produce Millimolar Concentrations of Non-Neuronal Acetylcholine for Breeding: Possible Adverse Effects of Neonicotinoids.

    Directory of Open Access Journals (Sweden)

    Ignaz Wessler

    Full Text Available The worldwide use of neonicotinoid pesticides has caused concern on account of their involvement in the decline of bee populations, which are key pollinators in most ecosystems. Here we describe a role of non-neuronal acetylcholine (ACh for breeding of Apis mellifera carnica and a so far unknown effect of neonicotinoids on non-target insects. Royal jelly or larval food are produced by the hypopharyngeal gland of nursing bees and contain unusually high ACh concentrations (4-8 mM. ACh is extremely well conserved in royal jelly or brood food because of the acidic pH of 4.0. This condition protects ACh from degradation thus ensuring delivery of intact ACh to larvae. Raising the pH to ≥5.5 and applying cholinesterase reduced the content of ACh substantially (by 75-90% in larval food. When this manipulated brood was tested in artificial larval breeding experiments, the survival rate was higher with food supplemented by 100% with ACh (6 mM than with food not supplemented with ACh. ACh release from the hypopharyngeal gland and its content in brood food declined by 80%, when honeybee colonies were exposed for 4 weeks to high concentrations of the neonicotinoids clothianidin (100 parts per billion [ppb] or thiacloprid (8,800 ppb. Under these conditions the secretory cells of the gland were markedly damaged and brood development was severely compromised. Even field-relevant low concentrations of thiacloprid (200 ppb or clothianidin (1 and 10 ppb reduced ACh level in the brood food and showed initial adverse effects on brood development. Our findings indicate a hitherto unknown target of neonicotinoids to induce adverse effects on non-neuronal ACh which should be considered when re-assessing the environmental risks of these compounds. To our knowledge this is a new biological mechanism, and we suggest that, in addition to their well documented neurotoxic effects, neonicotinoids may contribute to honeybee colony losses consecutive to a reduction of the ACh

  18. Expression of five acetylcholine receptor subunit genes in Brugia malayi adult worms

    Directory of Open Access Journals (Sweden)

    Ben-Wen Li

    2015-12-01

    Full Text Available Acetylcholine receptors (AChRs are required for body movement in parasitic nematodes and are targets of “classical” anthelmintic drugs such as levamisole and pyrantel and of newer drugs such as tribendimidine and derquantel. While neurotransmission explains the effects of these drugs on nematode movement, their effects on parasite reproduction are unexplained. The levamisole AChR type (L-AChRs in Caenorhabditis elegans is comprised of five subunits: Cel-UNC-29, Cel-UNC-38, Cel-UNC-63, Cel-LEV-1 and Cel-LEV-8. The genome of the filarial parasite Brugia malayi contains nine AChRs subunits including orthologues of Cel-unc-29, Cel-unc-38, and Cel-unc-63. We performed in situ hybridization with RNA probes to localize the expression of five AChR genes (Bm1_35890-Bma-unc-29, Bm1_20330-Bma-unc-38, Bm1_38195-Bma-unc-63, Bm1_48815-Bma-acr-26 and Bm1_40515-Bma-acr-12 in B. malayi adult worms. Four of these genes had similar expression patterns with signals in body muscle, developing embryos, spermatogonia, uterine wall adjacent to stretched microfilariae, wall of Vas deferens, and lateral cord. Three L-AChR subunit genes (Bma-unc-29, Bma-unc-38 and Bma-unc-63 were expressed in body muscle, which is a known target of levamisole. Bma-acr-12 was co-expressed with these levamisole subunit genes in muscle, and this suggests that its protein product may form receptors with other alpha subunits. Bma-acr-26 was expressed in male muscle but not in female muscle. Strong expression signals of these genes in early embryos and gametes in uterus and testis suggest that AChRs may have a role in nervous system development of embryogenesis and spermatogenesis. This would be consistent with embryotoxic effects of drugs that target these receptors in filarial worms. Our data show that the expression of these receptor genes is tightly regulated with regard to localization in adult worms and developmental stage in embryos and gametes. These results may help to explain the

  19. Effect of purines on calcium-independent acetylcholine release at the mouse neuromuscular junction.

    Science.gov (United States)

    Veggetti, M; Muchnik, S; Losavio, A

    2008-07-17

    At the mouse neuromuscular junction, activation of adenosine A(1) and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca(2+) influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca(2+)-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) and the P2Y(12-13) agonist 2-methylthio-adenosine 5'-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A(1) and P2Y(12-13) receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5'-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran

  20. CB1 receptor antagonism increases hippocampal acetylcholine release: site and mechanism of action.

    Science.gov (United States)

    Degroot, Aldemar; Köfalvi, Attila; Wade, Mark R; Davis, Richard J; Rodrigues, Ricardo J; Rebola, Nelson; Cunha, Rodrigo A; Nomikos, George G

    2006-10-01

    Evidence indicates that blockade of cannabinoid receptors increases acetylcholine (ACh) release in brain cortical regions. Although it is assumed that this type of effect is mediated through CB1 receptor (CB1R) antagonism, several in vitro functional studies recently have suggested non-CB1R involvement. In addition, neither the precise neuroanatomical site nor the exact mechanisms underlying this effect are known. We thoroughly examined these issues using a combination of systemic and local administration of CB1R antagonists, different methods of in vivo microdialysis, CB1R knockout (KO) mice, tissue measurements of ACh, and immunochemistry. First, we showed that systemic injections of the CB1R antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR-141716A) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) dose-dependently increased hippocampal ACh efflux. Likewise, local hippocampal, but not septal, infusions of SR141716A or AM251 increased hippocampal ACh release. It is noteworthy that the stimulatory effects of systemically administered CB1R antagonists on hippocampal ACh release were completely abolished in CB1R KO mice. CB1R KO mice had similar basal but higher stress-enhanced hippocampal ACh levels compared with wild-type controls. It is interesting that dopamine D1 receptor antagonism counteracted the stimulatory effect of CB1R blockade on hippocampal ACh levels. Finally, immunohistochemical methods revealed that a high proportion of CB1R-positive nerve terminals were found in hippocampus and confirmed the colocalization of CB1 receptors with cholinergic and dopaminergic nerve terminals. In conclusion, hippocampal ACh release may specifically be controlled through CB1Rs located on both cholinergic and dopaminergic neuronal projections, and CB1R antagonism increases hippocampal ACh release, probably through both a direct

  1. Acetylcholine versus cold pressor testing for evaluation of coronary endothelial function.

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    Ahmed AlBadri

    Full Text Available Assessment of coronary endothelial function with intracoronary acetylcholine (IC-Ach provides diagnostic and prognostic data in patients with suspected coronary microvascular dysfunction (CMD, but is often not feasible due in part to the time and expertise needed for pharmacologic mixing. Cold pressor testing (CPT is a simple and safe stimulus useful for either invasive or non-invasive endothelial function testing and myocardial perfusion imaging but has not been specifically evaluated among symptomatic women with signs of ischemic heart disease (IHD who have no obstructive coronary artery disease (CAD.163 women with signs and symptoms of IHD and no obstructive CAD from the NHLBI- Women's Ischemia Syndrome Evaluation-Coronary Vascular Dysfunction (WISE-CVD study underwent coronary reactivity testing with a Doppler flow wire (FloWire® Volcano, San Diego, CA in the proximal left anterior descending artery. Coronary artery diameter and coronary blood flow (CBF assessed by core lab using QCA before and after IC-Ach (18.2 μg/ml infused over 3 minutes and during CPT.Mean age was 55 ± 12 years. Rate pressure product (RPP in response to IC-Ach did not change (baseline to peak, P = 0.26, but increased during CPT (363±1457; P = 0.0028. CBF in response to CPT was poorly correlated to IC-Ach CBF. Change in coronary artery diameter after IC-Ach correlated with change after CPT (r = 0.59, P<0.001. The correlation coefficient was stronger in subjects with coronary dilation to IC-Ach (r = 0.628, P<0.001 versus those without dilation (r = 0.353, P = 0.002, suggesting that other factors may be important to this relationship when endothelium is abnormal.In women with no obstructive CAD and suspected CMD, coronary diameter changes with IC-Ach and CPT are moderately-well correlated suggesting that CPT testing may be of some use, particularly among patients with normal endothelial function, however, not an alternative to IC-Ach for diagnosis of coronary

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

    International Nuclear Information System (INIS)

    Welch, Kevin D.; Pfister, James A.; Lima, Flavia G.; Green, Benedict T.; Gardner, Dale R.

    2013-01-01

    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.

  3. Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

    Science.gov (United States)

    Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

    2015-10-01

    The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the

  4. Extracellular matrix organization in developing muscle: correlation with acetylcholine receptor aggregates.

    Science.gov (United States)

    Bayne, E K; Anderson, M J; Fambrough, D M

    1984-10-01

    Monoclonal antibodies recognizing laminin, heparan sulfate proteoglycan, fibronectin, and two apparently novel connective tissue components have been used to examine the organization of extracellular matrix of skeletal muscle in vivo and in vitro. Four of the five monoclonal antibodies are described for the first time here. Immunocytochemical experiments with frozen-sectioned muscle demonstrated that both the heparan sulfate proteoglycan and laminin exhibited staining patterns identical to that expected for components of the basal lamina. In contrast, the remaining matrix constituents were detected in all regions of muscle connective tissue: the endomysium, perimysium, and epimysium. Embryonic muscle cells developing in culture elaborated an extracellular matrix, each antigen exhibiting a unique distribution. Of particular interest was the organization of extracellular matrix on myotubes: the build-up of matrix components was most apparent in plaques overlying clusters of an integral membrane protein, the acetylcholine receptor (AChR). The heparan sulfate proteoglycan was concentrated at virtually all AChR clusters and showed a remarkable level of congruence with receptor organization; laminin was detected at 70-95% of AChR clusters but often was not completely co-distributed with AChR within the cluster; fibronectin and the two other extracellular matrix antigens occurred at approximately 20, 8, and 2% of the AChR clusters, respectively, and showed little or no congruence with AChR. From observations on the distribution of extracellular matrix components in tissue cultured fibroblasts and myogenic cells, several ideas about the organization of extracellular matrix are suggested. (a) Congruence between AChR clusters and heparan sulfate proteoglycan suggests the existence of some linkage between the two molecules, possibly important for regulation of AChR distribution within the muscle membrane. (b) The qualitatively different patterns of extracellular matrix

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

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    Gigout Sylvain

    2012-04-01

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

  6. Mice Lacking the Alpha9 Subunit of the Nicotinic Acetylcholine Receptor Exhibit Deficits in Frequency Difference Limens and Sound Localization

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    Amanda Clause

    2017-06-01

    Full Text Available Sound processing in the cochlea is modulated by cholinergic efferent axons arising from medial olivocochlear neurons in the brainstem. These axons contact outer hair cells in the mature cochlea and inner hair cells during development and activate nicotinic acetylcholine receptors composed of α9 and α10 subunits. The α9 subunit is necessary for mediating the effects of acetylcholine on hair cells as genetic deletion of the α9 subunit results in functional cholinergic de-efferentation of the cochlea. Cholinergic modulation of spontaneous cochlear activity before hearing onset is important for the maturation of central auditory circuits. In α9KO mice, the developmental refinement of inhibitory afferents to the lateral superior olive is disturbed, resulting in decreased tonotopic organization of this sound localization nucleus. In this study, we used behavioral tests to investigate whether the circuit anomalies in α9KO mice correlate with sound localization or sound frequency processing. Using a conditioned lick suppression task to measure sound localization, we found that three out of four α9KO mice showed impaired minimum audible angles. Using a prepulse inhibition of the acoustic startle response paradigm, we found that the ability of α9KO mice to detect sound frequency changes was impaired, whereas their ability to detect sound intensity changes was not. These results demonstrate that cholinergic, nicotinic α9 subunit mediated transmission in the developing cochlear plays an important role in the maturation of hearing.

  7. Gamma irradiation induces acetylcholine-evoked, endothelium-independent relaxation and activatesk-channels of isolated pulmonary artery of rats

    International Nuclear Information System (INIS)

    Eder, Veronique; Gautier, Mathieu; Boissiere, Julien; Girardin, Catherine; Rebocho, Manuel; Bonnet, Pierre

    2004-01-01

    Purpose: To test the effects of irradiation (R*) on the pulmonary artery (PA). Methods and materials: Isolated PA rings were submitted to gamma irradiation (cesium, 8 Gy/min -1 ) at doses of 20 Gy-140 Gy. Rings were placed in an organ chamber, contracted with serotonin (10 -4 M 5-hydroxytryptamine [5-HT]), then exposed to acetylcholine (ACh) in incremental concentrations. Smooth muscle cell (SMC) membrane potential was measured with microelectrodes. Results: A high dose of irradiation (60 Gy) increased 5HT contraction by 20%, whereas lower (20 Gy) doses slightly decreased it compared with control. In the absence of the endothelium, 5-HT precontracted rings exposed to 20 Gy irradiation developed a dose-dependent relaxation induced by acetylcholine (EI-ACh) with maximal relaxation of 60 ± 17% (n = 13). This was totally blocked by L-NAME (10 -4 M), partly by 7-nitro indazole; it was abolished by hypoxia and iberiotoxin, decreased by tetra-ethyl-ammonium, and not affected by free radical scavengers. In irradiated rings, hypoxia induced a slight contraction which was never observed in control rings. No differences in SMC membrane potential were observed between irradiated and nonirradiated PA rings. Conclusion: Irradiation mediates endothelium independent relaxation by a mechanism involving the nitric oxide pathway and K-channels

  8. Activation and modulation of human α4β2 nicotinic acetylcholine receptors by the neonicotinoids clothianidin and imidacloprid.

    Science.gov (United States)

    Li, Ping; Ann, Jason; Akk, Gustav

    2011-08-01

    Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. Though highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1-4% of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI for the human α4β2 receptor is 18 μM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)(2) (β2)(3) receptors significantly more strongly inhibited than the (α4)(3) (β2)(2) receptors. Copyright © 2011 Wiley-Liss, Inc.

  9. Fabrication of a Highly-sensitive Acetylcholine Sensor Based on AChOx Immobilized Smart-chips

    Directory of Open Access Journals (Sweden)

    M. M. RAHMAN

    2011-03-01

    Full Text Available Acetylcholine (ACh sensor based on acetylcholine oxidase (AChOx on EDC activated thioglycolic acid self-assembled monolayer (TGA-SAM using smart-chip has been developed. The simple cyclic voltammetry (CV, at 0.1 V/s technique is performed in total investigation, where 0.5M K3Fe(CN6 is utilized as a standard mediator in phosphate buffer solution (PBS, 0.1M. The ACh sensor exhibited a lower detection limit (DL, 0.1392 ± 0.005 nM, a wide linear dynamic range (LDR, 1.0 nM to 1.0 mM, good linearity (R=0.9951, and higher sensitivity (7.3543 ± 0.2 μAμM-1cm-2, and required small sample volume (70.0 μL as well as good stability and reproducibility. The smart-chip system employed a simple and efficient approach to the immobilization of enzymes onto active sensitive surface, which can enhance sensor performances to a large group of bio-molecules for wide range of biomedical applications in health care fields.

  10. Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

    Science.gov (United States)

    Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

    2017-06-05

    Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  11. In-vivo measurements of regional acetylcholine esterase activity in degenerative dementia: comparison with blood flow and glucose metabolism.

    Science.gov (United States)

    Herholz, K; Bauer, B; Wienhard, K; Kracht, L; Mielke, R; Lenz, M O; Strotmann, T; Heiss, W D

    2000-01-01

    Memory and attention are cognitive functions that depend heavily on the cholinergic system. Local activity of acetylcholine esterase (AChE) is an indicator of its integrity. Using a recently developed tracer for positron emission tomography (PET), C-11-labeled N-methyl-4-piperidyl-acetate (C11-MP4A), we measured regional AChE activity in 4 non-demented subjects, 4 patients with dementia of Alzheimer type (DAT) and 1 patient with senile dementia of Lewy body type (SDLT), and compared the findings with measurements of blood flow (CBF) and glucose metabolism (CMRGlc). Initial tracer extraction was closely related to CBF. AChE activity was reduced significantly in all brain regions in demented subjects, whereas reduction of CMRGlc and CBF was more limited to temporo-parietal association areas. AChE activity in SDLT was in the lower range of values in DAT. Our results indicate that, compared to non-demented controls, there is a global reduction of cortical AChE activity in dementia. Dementia, cholinergic system, acetylcholine esterase, positron emission tomography, cerebral blood flow, cerebral glucose metabolism.

  12. Isotopic rubidium ion efflux assay for the functional characterization of nicotinic acetylcholine receptors on clonal cell lines

    International Nuclear Information System (INIS)

    Lukas, R.J.; Cullen, M.J.

    1988-01-01

    An isotopic rubidium ion efflux assay has been developed for the functional characterization of nicotinic acetylcholine receptors on cultured neurons. This assay first involves the intracellular sequestration of isotopic potassium ion analog by the ouabain-sensitive action of a sodium-potassium ATPase. Subsequently, the release of isotopic rubidium ion through nicotinic acetylcholine receptor-coupled monovalent cation channels is activated by application of nicotinic agonists. Specificity of receptor-mediated efflux is demonstrated by its sensitivity to blockade by nicotinic, but not muscarinic, antagonists. The time course of agonist-mediated efflux, within the temporal limitations of the assay, indicates a slow inactivation of receptor function on prolonged exposure to agonist. Dose-response profiles (i) have characteristic shapes for different nicotinic agonists, (ii) are described by three operationally defined parameters, and (iii) reflect different affinities of agonists for binding sites that control receptor activation and functional inhibition. The rubidium ion efflux assay provides fewer hazards but greater sensitivity and resolution than isotopic sodium or rubidium ion influx assays for functional nicotinic receptors

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

    International Nuclear Information System (INIS)

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

    1981-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Directory of Open Access Journals (Sweden)

    Douglas A Schober

    Full Text Available Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S. Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  16. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    Science.gov (United States)

    Schober, Douglas A; Croy, Carrie H; Ruble, Cara L; Tao, Ran; Felder, Christian C

    2017-01-01

    Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  17. Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Delta2 -isoxazoline derivatives as potent and selective agonists of alpha7 nicotinic acetylcholine receptors

    DEFF Research Database (Denmark)

    Dallanoce, Clelia; Magrone, Pietro; Matera, Carlo

    2011-01-01

    A set of racemic spirocyclic quinuclidinyl-¿(2) -isoxazoline derivatives was synthesized using a 1,3-dipolar cycloaddition-based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (a7) and heteromeric (a4ß2) nicotinic acetylcholine receptors. ¿(2) -Isoxazol...

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

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    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...

  19. Positive allosteric modulators of the α7 nicotinic acetylcholine receptor potentiate glutamate release in the prefrontal cortex of freely-moving rats

    DEFF Research Database (Denmark)

    Bortz, D M; Upton, B A; Mikkelsen, J D

    2016-01-01

    Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptors (α7nAChRs) exhibit pro-cognitive effects in animal models of schizophrenia and are targets for the discovery of cognition-enhancing drugs. However, little is known about their in vivo mechanism of action because...

  20. Dual Nicotinic Acetylcholine Receptor α4β2 Antagonists/α7 Agonists: Synthesis, Docking Studies, and Pharmacological Evaluation of Tetrahydroisoquinolines and Tetrahydroisoquinolinium Salts

    DEFF Research Database (Denmark)

    Crestey, François; Jensen, Anders A; Soerensen, Christian

    2018-01-01

    We describe the synthesis of tetrahydroisoquinolines and tetrahydroisoquinolinium salts together with their pharmacological properties at various nicotinic acetylcholine receptors. In general, the compounds were α4β2 nAChR antagonists, with the tetrahydroisoquinolinium salts being more potent than...

  1. Structural differences in the two agonist binding sites of the Torpedo nicotinic acetylcholine receptor revealed by time-resolved fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Martinez, K. L.; Corringer, P. J.; Edelstein, S. J.

    2000-01-01

    The nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata carries two nonequivalent agonist binding sites at the αδ and αγ subunit interfaces. These sites have been characterized by time-resolved fluorescence with the partial nicotinic agonist dansyl-C6-choline (Dnscho). When bound...

  2. A combined α7 nicotinic acetylcholine receptor agonist and monoamine reuptake inhibitor, NS9775, represents a novel profile with potential benefits in emotional and cognitive disturbances

    DEFF Research Database (Denmark)

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

    2013-01-01

    also improve by nicotinic acetylcholine receptor (nAChR) activation. Preclinical studies have corroborated this and also demonstrated a synergistic antidepressant-like action when nAChR agonists and MRIs are combined. Here, we present the in vitro and in vivo profile of NS9775, a combined full α7 n...

  3. The nicotinic alpha7 acetylcholine receptor agonist ssr180711 is unable to activate limbic neurons in mice overexpressing human amyloid-beta1-42

    DEFF Research Database (Denmark)

    Soderman, A.; Spang-Thomsen, Mogens; Hansen, H.

    2008-01-01

    Recent studies have demonstrated that amyloid-beta1-42 (Abeta1-42) binds to the nicotinergic alpha7 acetylcholine receptor (alpha7 nAChR) and that the application of Abeta1-42 to cells inhibits the function of the alpha7 nAChR. The in vivo consequences of the pharmacological activation of the alp...

  4. Repeated potentiation of the metabotropic glutamate receptor 5 and the alpha 7 nicotinic acetylcholine receptor modulates behavioural and GABAergic deficits induced by early postnatal phencyclidine (PCP) treatment

    DEFF Research Database (Denmark)

    Kjaerby, Celia; Bundgaard, Christoffer; Fejgin, Kim

    2013-01-01

    GluR5), ADX47273, and the partial agonist of the α7 nicotinic acetylcholine receptor (α7 nAChR), SSR180711. Adolescent rats (4-5 weeks) subjected to PCP treatment during the second postnatal week displayed a consistent deficit in prepulse inhibition (PPI), which was reversed by a one-week treatment...

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

    DEFF Research Database (Denmark)

    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 vitro...

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

    DEFF Research Database (Denmark)

    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 (5...

  7. Role of the cholinergic nervous system in rheumatoid arthritis: aggravation of arthritis in nicotinic acetylcholine receptor alpha7 subunit gene knockout mice

    NARCIS (Netherlands)

    van Maanen, Marjolein A.; Stoof, Susanne P.; Larosa, Gregory J.; Vervoordeldonk, Margriet J.; Tak, Paul P.

    2010-01-01

    BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis

  8. Early postdenervation depolarization develops faster at endplates of hibernating golden hamsters where spontaneous quantal and non-quantal acetylcholine release is very small

    Czech Academy of Sciences Publication Activity Database

    Moravec, J.; Vyskočil, František

    2005-01-01

    Roč. 51, č. 1 (2005), s. 25-29 ISSN 0168-0102 R&D Projects: GA AV ČR IAA5011411; GA ČR GA305/02/1333 Institutional research plan: CEZ:AV0Z5011922 Keywords : motor nerve ending * non-quantal * acetylcholine Subject RIV: ED - Physiology Impact factor: 2.184, year: 2005

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

    NARCIS (Netherlands)

    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

  10. Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Vulfius, C.A.; Corbacheva, E.V.; Mordvintsev, D.Y.; Utkin, Y.N.; van Elk, R.; Smit, A.B.; Tsetlin, V.I.

    2006-01-01

    α-Conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of α-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into α-conotoxins

  11. Presynaptic inhibition of spontaneous acetylcholine release induced by adenosine at the mouse neuromuscular junction.

    Science.gov (United States)

    De Lorenzo, Silvana; Veggetti, Mariela; Muchnik, Salomón; Losavio, Adriana

    2004-05-01

    1. At the mouse neuromuscular junction, adenosine (AD) and the A(1) agonist 2-chloro-N(6)-cyclopentyl-adenosine (CCPA) induce presynaptic inhibition of spontaneous acetylcholine (ACh) release by activation of A(1) AD receptors through a mechanism that is still unknown. To evaluate whether the inhibition is mediated by modulation of the voltage-dependent calcium channels (VDCCs) associated with tonic secretion (L- and N-type VDCCs), we measured the miniature end-plate potential (mepp) frequency in mouse diaphragm muscles. 2. Blockade of VDCCs by Cd(2+) prevented the effect of the CCPA. Nitrendipine (an L-type VDCC antagonist) but not omega-conotoxin GVIA (an N-type VDCC antagonist) blocked the action of CCPA, suggesting that the decrease in spontaneous mepp frequency by CCPA is associated with an action on L-type VDCCs only. 3. As A(1) receptors are coupled to a G(i/o) protein, we investigated whether the inhibition of PKA or the activation of PKC is involved in the presynaptic inhibition mechanism. Neither N-(2[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89, a PKA inhibitor), nor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H-7, a PKC antagonist), nor phorbol 12-myristate 13-acetate (PHA, a PKC activator) modified CCPA-induced presynaptic inhibition, suggesting that these second messenger pathways are not involved. 4. The effect of CCPA was eliminated by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester epsilon6TDelta-BM, which suggests that the action of CCPA to modulate L-type VDCCs may involve Ca(2+)-calmodulin. 5. To investigate the action of CCPA on diverse degrees of nerve terminal depolarization, we studied its effect at different external K(+) concentrations. The effect of CCPA on ACh secretion evoked by 10 mm K(+) was prevented by the P/Q-type VDCC antagonist omega-agatoxin IVA. 6. CCPA failed to

  12. Cognitive correlates of α4β2 nicotinic acetylcholine receptors in mild Alzheimer's dementia.

    Science.gov (United States)

    Sabri, Osama; Meyer, Philipp M; Gräf, Susanne; Hesse, Swen; Wilke, Stephan; Becker, Georg-Alexander; Rullmann, Michael; Patt, Marianne; Luthardt, Julia; Wagenknecht, Gudrun; Hoepping, Alexander; Smits, Rene; Franke, Annegret; Sattler, Bernhard; Tiepolt, Solveig; Fischer, Steffen; Deuther-Conrad, Winnie; Hegerl, Ulrich; Barthel, Henryk; Schönknecht, Peter; Brust, Peter

    2018-06-01

    In early Alzheimer's dementia, there is a need for PET biomarkers of disease progression with close associations to cognitive dysfunction that may aid to predict further cognitive decline and neurodegeneration. Amyloid biomarkers are not suitable for that purpose. The α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) are widely abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as attention, learning and memory. Post-mortem studies reported lower expression of α4β2-nAChRs in more advanced Alzheimer's dementia. However, there is ongoing controversy whether α4β2-nAChRs are reduced in early Alzheimer's dementia. Therefore, using the recently developed α4β2-nAChR-specific radioligand (-)-18F-flubatine and PET, we aimed to quantify the α4β2-nAChR availability and its relationship to specific cognitive dysfunction in mild Alzheimer's dementia. Fourteen non-smoking patients with mild Alzheimer's dementia, drug-naïve for cholinesterase therapy, were compared with 15 non-smoking healthy controls matched for age, sex and education by applying (-)-18F-flubatine PET together with a neuropsychological test battery. The one-tissue compartment model and Logan plot method with arterial input function were used for kinetic analysis to obtain the total distribution volume (VT) as the primary, and the specific binding part of the distribution volume (VS) as the secondary quantitative outcome measure of α4β2-nAChR availability. VS was determined by using a pseudo-reference region. Correlations between VT within relevant brain regions and Z-scores of five cognitive functions (episodic memory, executive function/working memory, attention, language, visuospatial function) were calculated. VT (and VS) were applied for between-group comparisons. Volume of interest and statistical parametric mapping analyses were carried out. Analyses revealed that in patients with mild Alzheimer's dementia compared to healthy controls

  13. Effect of donepezil hydrochloride (E2020) on basal concentration of extracellular acetylcholine in the hippocampus of rats.

    Science.gov (United States)

    Kosasa, T; Kuriya, Y; Matsui, K; Yamanishi, Y

    1999-09-10

    The effects of oral administration of the centrally acting acetylcholinesterase (AChE) inhibitors, donepezil hydrochloride (donepezil: E2020: (+/-)-2-[(1-benzylpiperidin-4-yl)methyl]-5,6-dimethoxy-indan-1-one monohydrochloride), tacrine (9-amino-1,2,3,4-tetrahydroacridine hydrochloride) and ENA-713 (rivastigmine: (S)-N-ethyl-3-[(1-dimethyl-amino)ethyl]-N-methyl-phenylcarbamate hydrogentartrate), which have been developed for the treatment of Alzheimer's disease, on the extracellular acetylcholine concentration in the hippocampus of rats were evaluated by using a microdialysis technique without adding cholinesterase inhibitor to the perfusion solution. We also compared the inhibition of brain AChE and the brain concentrations of these drugs. Donepezil at 2.5 mg/kg and tacrine at 5 mg/kg showed significant effects for more than 6 h. At these doses, the maximum increases were observed at about 1.5 h after administration of donepezil, and at about 2 h with tacrine, and were 499% and 422% of the pre-level, respectively. ENA-713 produced significant effects at doses of 0.625, 1.25 and 2.5 mg/kg, which lasted for about 1, 2 and 4 h, respectively. The maximum increases produced by these doses at about 0.5 h after administration were 190, 346 and 458% of the pre-level, respectively. The time courses of brain AChE inhibition with donepezil at 2.5 mg/kg, tacrine at 10 mg/kg and ENA-713 at 2.5 mg/kg were mirror images of the extracellular acetylcholine-increasing action at the same doses. The time courses of the brain concentrations of drugs after oral administration of donepezil at 2.5 mg/kg and tacrine at 10 mg/kg were consistent with those of brain AChE inhibition at the same doses, and there was a linear relation between these parameters. Brain concentration of ENA-713 at 2.5 mg/kg was below the limit of quantification at all time points measured. These results suggest that oral administration of donepezil, tacrine and ENA-713 increases acetylcholine concentration in the

  14. Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant

    Directory of Open Access Journals (Sweden)

    Irina V. Shelukhina

    2018-01-01

    Full Text Available Azemiopsin (Az, a linear peptide from the Azemiops feae viper venom, contains no disulfide bonds, is a high-affinity and selective inhibitor of nicotinic acetylcholine receptor (nAChR of muscle type and may be considered as potentially applicable nondepolarizing muscle relaxant. In this study, we investigated its preclinical profile in regard to in vitro and in vivo efficacy, acute and chronic toxicity, pharmacokinetics, allergenic capacity, immunotoxicity and mutagenic potency. The peptide effectively inhibited (IC50 ~ 19 nM calcium response of muscle nAChR evoked by 30 μM (EC100 acetylcholine but was less potent (IC50 ~ 3 μM at α7 nAChR activated by 10 μM (EC50 acetylcholine and had a low affinity to α4β2 and α3-containing nAChR, as well as to GABAA or 5HT3 receptors. Its muscle relaxant effect was demonstrated at intramuscular injection to mice at doses of 30–300 µg/kg, 30 µg/kg being the initial effective dose and 90 µg/kg—the average effective dose. The maximal muscle relaxant effect of Az was achieved in 10 min after the administration and elimination half-life of Az in mice was calculated as 20–40 min. The longest period of Az action observed at a dose of 300 µg/kg was 55 min. The highest acute toxicity (LD50 510 μg/kg was observed at intravenous injection of Az, at intramuscular or intraperitoneal administration it was less toxic. The peptide showed practically no immunotoxic, allergenic or mutagenic capacity. Overall, the results demonstrate that Az has good drug-like properties for the application as local muscle relaxant and in its parameters, is not inferior to the relaxants currently used. However, some Az modification might be effective to extend its narrow therapeutic window, a typical characteristic and a weak point of all nondepolarizing myorelaxants.

  15. Acetylcholine-Binding Protein Engineered to Mimic the α4-α4 Binding Pocket in α4β2 Nicotinic Acetylcholine Receptors Reveals Interface Specific Interactions Important for Binding and Activity

    DEFF Research Database (Denmark)

    Shahsavar, Azadeh; Ahring, Philip K; Olsen, Jeppe A

    2015-01-01

    Neuronal α4β2 nicotinic acetylcholine receptors are attractive drug targets for psychiatric and neurodegenerative disorders and smoking cessation aids. Recently, a third agonist binding site between two α4 subunits in the (α4)(3)(β2)(2) receptor subpopulation was discovered. In particular, three......-yl)-1,4-diazepane], highlights the roles of the three residues in determining binding affinities and functional properties of ligands at the α4-α4 interface. Confirmed by mutational studies, our structures suggest a unique ligand-specific role of residue H142 on the α4 subunit. In the cocrystal...... that could not be predicted based on wild-type Ls-AChBP structures in complex with the same agonists. The results show that an unprecedented correlation between binding in engineered AChBPs and functional receptors can be obtained and provide new opportunities for structure-based design of drugs targeting...

  16. Supplementing female rats with DHA-lysophosphatidylcholine increases docosahexaenoic acid and acetylcholine contents in the brain and improves the memory and learning capabilities of the pups

    Energy Technology Data Exchange (ETDEWEB)

    Valenzuela, A.; Nieto, S.; Sanhueza, J.; Morgado, N.; Rojas, I.; Zanartu, P.

    2010-07-01

    Docosahexaenoic acid (Dha) is supplied to the foetus and newborn through the mother from their own reserves and their diet. No consensus about the best form to supplement DHA has been established. We propose that DHA containing lysophosphatidylcholine (DHA-LPC), obtained from DHA-rich eggs may be a suitable form of DHA and choline (the precursor of acetylcholine) supplementation. We evaluated the effectiveness of DHA-LPC to increase DHA and acetylcholine concentration in the brain of pups born from female rats supplemented with DHA-LPC before and during pregnancy. We also evaluated the effect of DHA supplementation on learning and memory capabilities of pups through the Skinner test for operant conditioning. Female Wistar rats received 40-day supplementation of DHA-LPC (8 mg DHA/kg b.w/daily.), before and during pregnancy. After delivery, plasma, erythrocyte, liver, and adipose tissue DHA and plasma choline were analyzed. Brains from 60 day-old pups separated into frontal cortex, cerebellum, striatum, hippocampus, and occipital cortex, were assessed for DHA, acetylcholine, and acetylcholine transferase (CAT) activity. Pups were subjected to the Skinner box test. DHA-LPC supplementation produces higher choline and liver DHA contents in the mothers plasma and increases the pups DHA and acetylcholine in the cerebellum and hippocampus. CAT was not modified by supplementation. The Skinner test shows that pups born from DHA-LPC supplemented mothers exhibit better scores of learning and memory than the controls. Conclusion: DHA-LPC may be an adequate form for DHA supplementation during the perinatal period. (Author) 66 refs.

  17. Flow- and acetylcholine-induced dilation in small arteries from rats with renovascular hypertension - effect of tempol treatment

    DEFF Research Database (Denmark)

    Christensen, Frank Holden; Stankevicius, Edgaras; Hansen, Thomas

    2007-01-01

    -treated animals, while only the relaxation was improved by the NO donor, S-nitroso-N-acetylpenicillamine (SNAP). In conclusion renovascular hypertension selectively inhibits flow-induced NO-mediated vasodilatation, while EDHF-type vasodilatation remains unaffected, suggesting that high blood pressure leads...... blood pressure and tempol treatment. Simultaneous measurements of NO-concentration and relaxation were performed in isolated coronary arteries from the same animals. As compared to vehicle-treated rats, both acetylcholine-induced relaxation and NO-concentration increased in arteries from tempol......-induced dilatation remained normal. Measured by dihydroethidium staining there was an increased amount of superoxide in arteries from vehicle-treated rats, but not from tempol-treated rats. Expression by immunoblotting of endothelial NO synthase and the NAD(P)H oxidase subunit p47phox remained unaffected by high...

  18. Safety and efficacy of eculizumab in anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis (REGAIN)

    DEFF Research Database (Denmark)

    Howard, James F; Utsugisawa, Kimiaki; Benatar, Michael

    2017-01-01

    BACKGROUND: Complement is likely to have a role in refractory generalised myasthenia gravis, but no approved therapies specifically target this system. Results from a phase 2 study suggested that eculizumab, a terminal complement inhibitor, produced clinically meaningful improvements in patients...... with anti-acetylcholine receptor antibody-positive refractory generalised myasthenia gravis. We further assessed the efficacy and safety of eculizumab in this patient population in a phase 3 trial. METHODS: We did a phase 3, randomised, double-blind, placebo-controlled, multicentre study (REGAIN) in 76...... hospitals and specialised clinics in 17 countries across North America, Latin America, Europe, and Asia. Eligible patients were aged at least 18 years, with a Myasthenia Gravis-Activities of Daily Living (MG-ADL) score of 6 or more, Myasthenia Gravis Foundation of America (MGFA) class II-IV disease...

  19. A comparison between endogenous acetylcholiner release and (3H) choline outflow from guinea-pig brain slices

    International Nuclear Information System (INIS)

    Beani, L.; Bianchi, C.; Siniscalchi, A.; Sivilotti, L.; Tanganelli, S.; Veratti, E.

    1986-01-01

    The measure of tritium-choline efflux from preloaded brain slices is considered a valid method for studying cholinergic functiton. At variance with th say of endogenous acetylcholine this procedure gives andindex of the release process in the absence of esterase inhibition, thus excluding the dampening of the autoreceptor-mediated negative feedback. In order to establish the equivalence of these approaches the tow methods have been compared on electrically=stimulated guinea-pig brain slices, kept under the ame experimental conditions. The results show that only a partial equivalence of the two methods can be recognized. A drawback of the tritium-choline approach is the exhaustion or dilution of tritium stores, so that drug-induced increases of evoked efflux are minimized

  20. Acetylcholine serves as a derepressor in Loperamide-induced Opioid-Induced Bowel Dysfunction (OIBD) in zebrafish.

    Science.gov (United States)

    Shi, Yanyan; Zhang, Yu; Zhao, Fangying; Ruan, Hua; Huang, Honghui; Luo, Lingfei; Li, Li

    2014-07-07

    The mechanisms underlying gut development, especially peristalsis, are widely studied topics. However, the causes of gut peristalsis-related diseases, especially Opioid-Induced Bowel Dysfunction (OIBD) disorder, have not been well defined. Therefore, our study used zebrafish, a popular model for studying both gut development and peristalsis, and DCFH-DA, a dye that clearly labels the live fish gut lumen, to characterize the formation process of gut lumen as well as the gut movement style in vivo. By applying Loperamide Hydrochloride (LH), the μ-opioid receptor-specific agonist, we established an OIBD-like zebrafish model. Our study found that acetylcholine (ACh) was a key transmitter that derepressed the phenotype induced by LH. Overall, the study showed that the antagonistic role of ACh in the LH-mediated opioid pathway was evolutionarily conserved; moreover, the OIBD-like zebrafish model will be helpful in the future dissection of the molecular pathways involved in gut lumen development and pathology.

  1. 6-[{sup 18}F]fluoro-A-85380: an in vivo tracer for the nicotinic acetylcholine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Scheffel, Ursula; Horti, Andrew G.; Koren, Andrei O.; Ravert, Hayden T.; Banta, Jeffrey P.; Finley, Paige A.; London, Edythe D.; Dannals, Robert F. E-mail: robert_dannals@tracer.nm.jhu.edu

    2000-01-01

    6-[{sup 18}F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[{sup 18}F]fluoro-A-85380 or 6-[{sup 18}F]FA), a new tracer for positron emission tomography, was synthesized by no-carrier-added [{sup 18}F] fluorination of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine followed by acidic deprotection. 6-[{sup 18}F]FA followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 6-FA. A preliminary toxicology study of the 6-FA showed a relatively low biological effect.

  2. 6-[18F]fluoro-A-85380: an in vivo tracer for the nicotinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Scheffel, Ursula; Horti, Andrew G.; Koren, Andrei O.; Ravert, Hayden T.; Banta, Jeffrey P.; Finley, Paige A.; London, Edythe D.; Dannals, Robert F.

    2000-01-01

    6-[ 18 F]Fluoro-3-(2(S)-azetidinylmethoxy)pyridine (6-[ 18 F]fluoro-A-85380 or 6-[ 18 F]FA), a new tracer for positron emission tomography, was synthesized by no-carrier-added [ 18 F] fluorination of 6-iodo-3-((1-tert-butoxycarbonyl-2(S)-azetidinyl)methoxy)pyridine followed by acidic deprotection. 6-[ 18 F]FA followed the regional densities of brain nicotinic acetylcholine receptors (nAChRs) reported in the literature. Evidence of binding to nAChRs and high specificity of the binding in vivo was demonstrated by inhibition with nAChR selective ligands as well as with unlabeled 6-FA. A preliminary toxicology study of the 6-FA showed a relatively low biological effect

  3. Exploring the binding energy profiles of full agonists, partial agonists, and antagonists of the α7 nicotinic acetylcholine receptor.

    Science.gov (United States)

    Tabassum, Nargis; Ma, Qianyun; Wu, Guanzhao; Jiang, Tao; Yu, Rilei

    2017-09-01

    Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

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

    DEFF Research Database (Denmark)

    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...... 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....

  5. Acetylcholine receptor binding antibody-associated myasthenia gravis and rhabdomyolysis induced by nivolumab in a patient with melanoma.

    Science.gov (United States)

    Shirai, Takushi; Sano, Tasuku; Kamijo, Fuminao; Saito, Nana; Miyake, Tomomi; Kodaira, Minori; Katoh, Nagaaki; Nishie, Kenichi; Okuyama, Ryuhei; Uhara, Hisashi

    2016-01-01

    We reported an 81-year-old woman with metastatic melanoma, in whom myasthenia gravis and rhabdomyolysis developed after nivolumab monotherapy. The first symptom of myasthenia gravis was dyspnea. Ultrasonography detected hypokinesis of the bilateral diaphragm suggesting myasthenia gravis, although there was no abnormal finding of the lungs in computed tomography images. Acetylcholine receptor binding antibodies were low-titer positive in the preserved serum before administration of nivolumab, strongly suggesting that the myasthenia gravis was a nivolumab-related immune adverse event. Despite the remarkable clinical benefits of immune checkpoint inhibitors for patients with advanced melanoma, it is important to recognize unexpected immune-related adverse events. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  6. Association of nicotinic acetylcholine receptors with central respiratory control in isolated brainstem-spinal cord preparation of neonatal rats

    Directory of Open Access Journals (Sweden)

    EIKI HATORI

    2006-01-01

    Full Text Available Nicotine exposure is a risk factor in several breathing disorders Nicotinic acetylcholine receptors (nAChRs exist in the ventrolateral medulla, an important site for respiratory control. We examined the effects of nicotinic acetylcholine neurotransmission on central respiratory control by addition of a nAChR agonist or one of various antagonists into superfusion medium in the isolated brainstem-spinal cord from neonatal rats. Ventral C4 neuronal activity was monitored as central respiratory output, and activities of respiratory neurons in the ventrolateral medulla were recorded in whole-cell configuration. RJR-2403 (0.1-10mM, alpha4beta2 nAChR agonist induced dose-dependent increases in respiratory frequency. Non-selective nAChR antagonist mecamylamine (0.1-100mM, alpha4beta2 antagonist dihydro-beta-erythroidine (0.1-100mM, alpha7 antagonist methyllycaconitine (0.1-100mM, and a-bungarotoxin (0.01-10mM all induced dose-dependent reductions in C4 respiratory rate. We next examined effects of 20mM dihydro-beta-erythroidine and 20mM methyllycaconitine on respiratory neurons. Dihydro-beta-erythroidine induces hyperpolarization and decreases intraburst firing frequency of inspiratory and preinspiratory neurons. In contrast, methyllycaconitine has no effect on the membrane potential of inspiratory neurons, but does decrease their intraburst firing frequency while inducing hyperpolarization and decreasing intraburst firing frequency in preinspiratory neurons. These findings indicate that alpha4beta2 nAChR is involved in both inspiratory and preinspiratory neurons, whereas alpha7 nAChR functions only in preinspiratory neurons to modulate C4 respiratory rate

  7. Iptakalim inhibits nicotinic acetylcholine receptor-mediated currents in dopamine neurons acutely dissociated from rat substantia nigra pars compacta.

    Science.gov (United States)

    Hu, J; DeChon, J; Yan, K C; Liu, Q; Hu, G; Wu, J

    2006-07-31

    Iptakalim hydrochloride, a novel cardiovascular ATP-sensitive K(+) (K(ATP)) channel opener, has shown remarkable antihypertensive and neuroprotective effects in a variety of studies using in vivo and in vitro preparations. We recently found that iptakalim blocked human alpha4-containing nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the human SH-EP1 cell line. In the present study, we examined the effects of iptakalim on several neurotransmitter-induced current responses in single DA neurons freshly dissociated from rat substantia nigra pars compacta (SNc), using perforated patch-clamp recordings combined with a U-tube rapid drug application. In identified DA neurons under voltage-clamp configuration, glutamate-, NMDA-, and GABA-induced currents were insensitive to co-application with iptakalim (100 microM), while whole-cell currents induced by ACh (1 mM+1 microM atropine) or an alpha4beta2 nicotinic acetylcholine receptors relatively selective agonist, RJR-2403 (300 microM), were eliminated by iptakalim. Iptakalim inhibited RJR-2403-induced current in a concentration-dependent manner, and reduced maximal RJR-2403-induced currents at the highest agonist concentration, suggesting a non-competitive block. In current-clamp mode, iptakalim failed to affect resting membrane potential and spontaneous action potential firing, but abolished RJR-2403-induced neuronal firing acceleration. Together, these results indicate that in dissociated SNc DA neurons, alpha4-containing nAChRs, rather than ionotropic glutamate receptors, GABA(A) receptors or perhaps K-ATP channels are the sensitive targets to mediate iptakalim's pharmacological roles.

  8. Analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution

    International Nuclear Information System (INIS)

    Wise, D.S.; Karlin, A.; Schoenborn, B.P.

    1979-01-01

    The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. In this paper the results of the use of low-angle neutron scattering to investigate the shape of the receptor-detergent complex and separately of its protein and detergent moieties are reported. By adjustment of the neutron-scattering density of the solvent with D 2 O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in 18% D 2 O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +- 1 A. The radius of gyration and molecular volume (305,000 A 3 ) of the receptor is inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) 150 x 50 A or an oblate cylinder, 25 x 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +- 40,000), the extent of Triton X-100 binding in the complex (0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4

  9. Hinge-deleted IgG4 blocker therapy for acetylcholine receptor myasthenia gravis in rhesus monkeys.

    Science.gov (United States)

    Losen, Mario; Labrijn, Aran F; van Kranen-Mastenbroek, Vivianne H; Janmaat, Maarten L; Haanstra, Krista G; Beurskens, Frank J; Vink, Tom; Jonker, Margreet; 't Hart, Bert A; Mané-Damas, Marina; Molenaar, Peter C; Martinez-Martinez, Pilar; van der Esch, Eline; Schuurman, Janine; de Baets, Marc H; Parren, Paul W H I

    2017-04-20

    Autoantibodies against ion channels are the cause of numerous neurologic autoimmune disorders. Frequently, such pathogenic autoantibodies have a restricted epitope-specificity. In such cases, competing antibody formats devoid of pathogenic effector functions (blocker antibodies) have the potential to treat disease by displacing autoantibodies from their target. Here, we have used a model of the neuromuscular autoimmune disease myasthenia gravis in rhesus monkeys (Macaca mulatta) to test the therapeutic potential of a new blocker antibody: MG was induced by passive transfer of pathogenic acetylcholine receptor-specific monoclonal antibody IgG1-637. The effect of the blocker antibody (IgG4Δhinge-637, the hinge-deleted IgG4 version of IgG1-637) was assessed using decrement measurements and single-fiber electromyography. Three daily doses of 1.7 mg/kg IgG1-637 (cumulative dose 5 mg/kg) induced impairment of neuromuscular transmission, as demonstrated by significantly increased jitter, synaptic transmission failures (blockings) and a decrease in the amplitude of the compound muscle action potentials during repeated stimulations (decrement), without showing overt symptoms of muscle weakness. Treatment with three daily doses of 10 mg/kg IgG4Δhinge-637 significantly reduced the IgG1-637-induced increase in jitter, blockings and decrement. Together, these results represent proof-of principle data for therapy of acetylcholine receptor-myasthenia gravis with a monovalent antibody format that blocks binding of pathogenic autoantibodies.

  10. Melatonin modulation of presynaptic nicotinic acetylcholine receptors located on short noradrenergic neurons of the rat vas deferens: a pharmacological characterization

    Directory of Open Access Journals (Sweden)

    Zago W.M.

    1999-01-01

    Full Text Available Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP = cytisine > nicotine > carbachol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response during the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllycaconitine inhibited the nicotine-induced response only at 21:00 h. Since a7 nicotinic acetylcholine receptors (nAChRs have low affinity for nicotine in binding assays, we suggest that a mixed population composed of a3ß4 - plus a7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity binding site, probably an a7 nAChR subtype.

  11. Inflammation-induced increase in nicotinic acetylcholine receptor current in cutaneous nociceptive DRG neurons from the adult rat.

    Science.gov (United States)

    Zhang, X-L; Albers, K M; Gold, M S

    2015-01-22

    The goals of the present study were to determine (1) the properties of the nicotinic acetylcholine receptor (nAChR) currents in rat cutaneous dorsal root ganglion (DRG) neurons; (2) the impact of nAChR activation on the excitability of cutaneous DRG neurons; and (3) the impact of inflammation on the density and distribution of nAChR currents among cutaneous DRG neurons. Whole-cell patch-clamp techniques were used to study retrogradely labeled DRG neurons from naïve and complete Freund's adjuvant inflamed rats. Nicotine-evoked currents were detectable in ∼70% of the cutaneous DRG neurons, where only one of two current types, fast or slow currents based on rates of activation and inactivation, was present in each neuron. The biophysical and pharmacological properties of the fast current were consistent with nAChRs containing an α7 subunit while those of the slow current were consistent with nAChRs containing α3/β4 subunits. The majority of small diameter neurons with fast current were IB4- while the majority of small diameter neurons with slow current were IB4+. Preincubation with nicotine (1 μM) produced a transient (1 min) depolarization and increase in the excitability of neurons with fast current and a decrease in the amplitude of capsaicin-evoked current in neurons with slow current. Inflammation increased the current density of both slow and fast currents in small diameter neurons and increased the percentage of neurons with the fast current. With the relatively selective distribution of nAChR currents in putative nociceptive cutaneous DRG neurons, our results suggest that the role of these receptors in inflammatory hyperalgesia is likely to be complex and dependent on the concentration and timing of acetylcholine release in the periphery. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Adenosine A₁ and A₂A receptor-mediated modulation of acetylcholine release in the mice neuromuscular junction.

    Science.gov (United States)

    Garcia, Neus; Priego, Mercedes; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Besalduch, Nuria; Lanuza, M Angel; Tomàs, Josep

    2013-07-01

    Immunocytochemistry shows that purinergic receptors (P1Rs) type A1 and A2A (A1 R and A2 A R, respectively) are present in the nerve endings at the P6 and P30 Levator auris longus (LAL) mouse neuromuscular junctions (NMJs). As described elsewhere, 25 μm adenosine reduces (50%) acetylcholine release in high Mg(2+) or d-tubocurarine paralysed muscle. We hypothesize that in more preserved neurotransmission machinery conditions (blocking the voltage-dependent sodium channel of the muscle cells with μ-conotoxin GIIIB) the physiological role of the P1Rs in the NMJ must be better observed. We found that the presence of a non-selective P1R agonist (adenosine) or antagonist (8-SPT) or selective modulators of A1 R or A2 A R subtypes (CCPA and DPCPX, or CGS-21680 and SCH-58261, respectively) does not result in any changes in the evoked release. However, P1Rs seem to be involved in spontaneous release (miniature endplate potentials MEPPs) because MEPP frequency is increased by non-selective block but decreased by non-selective stimulation, with A1 Rs playing the main role. We assayed the role of P1Rs in presynaptic short-term plasticity during imposed synaptic activity (40 Hz for 2 min of supramaximal stimuli). Depression is reduced by micromolar adenosine but increased by blocking P1Rs with 8-SPT. Synaptic depression is not affected by the presence of selective A1 R and A2 A R modulators, which suggests that both receptors need to collaborate. Thus, A1 R and A2 A R might have no real effect on neuromuscular transmission in resting conditions. However, these receptors can conserve resources by limiting spontaneous quantal leak of acetylcholine and may protect synaptic function by reducing the magnitude of depression during repetitive activity. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface

    DEFF Research Database (Denmark)

    Ahring, Philip K.; Olsen, Jeppe A.; Nielsen, Elsebet O.

    2015-01-01

    The nicotinic acetylcholine receptor alpha 4 beta 2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (alpha 4)(2)(beta 2)(3) and (alpha 4)(3)(beta 2)(2). While these are similar in many aspects, the (alpha 4)(3)(beta 2)(2) stoichiometry...... differs by harboring a third orthosteric acetylcholine binding site located at the alpha 4-alpha 4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known....... The present study was therefore aimed at determining binding affinities of nicotinic ligands to the alpha 4-alpha 4 interface. Given that epibatidine shows large functional potency differences at alpha 4-beta 2 vs. alpha 4-alpha 4 interfaces, biphasic binding properties would be expected at (alpha 4)(3)(beta...

  15. α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Zwart, Ruud; Ursu, Daniel

    2015-01-01

    The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present in the h......The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer's disease, it is critical to determine whether α7β2 nAChRs are present...

  16. An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis

    International Nuclear Information System (INIS)

    Carter, B.; Harrison, R.; Lunt, G.G.; Morris, H.; Savage-Marengo, T.; Stephenson, F.A.

    1981-01-01

    A reproducible radioimmunoassay procedure for the determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis is described and examined in detail. The assay combines features of a number of methods previously outlined and allows repeat determinations of antibody titre in a given myasthenic serum sample with coefficient of variation 6%. The mean +- standard deviation for normal human serum anti-acetylcholine receptor antibodies was found by this procedure to be 0.024 +- 0.033 nmol/l α-bungarotoxin binding sites whereas the range for myasthenic patients was 0-139.14 nmol/l with a mean value of 7.55 nmol/l α-bungarotoxin binding sites. (author)

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

    DEFF Research Database (Denmark)

    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...... on efficacy. The shared modulatory profile along with a binding site located in an extracellular subunit interface suggest that modulation via an agonist-like mechanism may be a common mechanism of action that potentially could apply to Cys loop receptors beyond the α4β2 nAChRs....... modulator that targets the α4-α4 interface of α4β2 nicotinic acetylcholine receptors (nAChRs). Together with homology modeling, mutational data, quantum mechanical calculations, and pharmacological studies on α4β2 nAChRs, the structure reveals a modulator binding mode that overlaps the α4-α4 interface...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    by their inefficient functional expression in vitro. In the present study we have characterized and compared the pharmacological properties displayed by α6β4 and α6β4β3 nicotinic acetylcholine receptors assembled in tsA201 cells from the classical α6/α3 chimera (C1) and two novel α6/α3 chimeras (C6F223L and C16F223L...... 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 receptors....

  19. Structural Characterization of the Putative Cholinergic Binding Region alpha(179-201) of the Nicotinic Acetylcholine Receptor. Part 1. Review and Experimental Design.

    Science.gov (United States)

    1993-04-01

    SUBJCT TERMS .. 15. NUMBER OF PAGES Nicotinic acetylcholine receptor FTIR 21 Vibrational spectroscopy Cholinergic 16. PRICE COOE Resonance raman 17...Wilson et al 1955). FMR spectroscopy measures the absorbance of infra-red rad iation, where as Raman spectroscopy measures inelastic scattering of...frequency is domrunated by that chromophore, then Raman scattering involving vibrations localized in that chromophore will be sharply enhanced(Cantor and

  20. Effect of acetylcholine receptors on the pain-related electrical activities in the hippocampal CA3 region of morphine-addicted rats

    OpenAIRE

    Li, Guan Zeng; Liu, Zhe Hui; Wei, XinYa; Zhao, Pan; Yang, Chun Xiao; Xu, Man Ying

    2015-01-01

    Objective(s): To determine the effect of acetylcholine (ACh), pilocarpine, and atropine on pain evoked responses of pain excited neurons (PEN) and pain inhibited neurons (PIN) in hippocampal CA3 region of morphine addicted rats. Materials and Methods: Female Wistar rats, weighing between 230-260 g were used in this study. Morphine addicted rats were generated by subcutaneous injection of increasing concentrations of morphine hydrochloride for six days. Trains of electrical impulses applied to...

  1. Bisphenol A, Dichlorodiphenyltrichloroethane (DDT) and Vinclozolin Affect ex-vivo Uterine Contraction in Rats via Uterotonin (Prostaglandin F2?, Acetylcholine and Oxytocin) Related Pathways

    OpenAIRE

    Salleh, Naguib; Giribabu, Nelli; Feng, Angeline Oh Mei; Myint, Kyaimon

    2015-01-01

    Bisphenol-A (BPA), dichrolodiphenyltrichloroethane (DDT) and vinclozolin were found able to induce abnormal uterine contraction. The mechanisms involved remains unclear. We hypothesized that the effect of these compounds were mediated via the uterotonin pathways. Therefore, in this study, effects of BPA, vinclozolin and DDT-only and in combination with uterotonins (PGF-2?, acetylcholine and oxytocin) on the force and pattern of uterine contraction were observed. Methods: Uteri were harvested ...

  2. Wash-Resistantly Bound Xanomeline Inhibits Acetylcholine Release by Persistent Activation of Presynaptic M2 and M4 Muscarinic Receptors in Rat Brain

    Czech Academy of Sciences Publication Activity Database

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

    2007-01-01

    Roč. 322, č. 1 (2007), s. 316-323 ISSN 0022-3565 R&D Projects: GA ČR(CZ) GA305/05/0452; GA MŠk(CZ) LC554 Grant - others:-(US) NS25743 Institutional research plan: CEZ:AV0Z50110509 Keywords : acetylcholine * xanomeline * muscarinic receptor Subject RIV: ED - Physiology Impact factor: 4.003, year: 2007

  3. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Grandič, Marjana [Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana (Slovenia); Aráoz, Romulo; Molgó, Jordi [CNRS, Institut de Neurobiologie Alfred Fessard, FRC 2118, Laboratoire de Neurobiologie et Développement, UPR 3294, F-91198 Gif-sur-Yvette Cedex (France); Turk, Tom; Sepčić, Kristina [Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana (Slovenia); Benoit, Evelyne [CNRS, Institut de Neurobiologie Alfred Fessard, FRC 2118, Laboratoire de Neurobiologie et Développement, UPR 3294, F-91198 Gif-sur-Yvette Cedex (France); Frangež, Robert, E-mail: robert.frangez@vf.uni-lj.si [Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, SI-1000 Ljubljana (Slovenia)

    2012-12-01

    APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC{sub 50} = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC{sub 50} = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1{sub 2}β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC{sub 50} = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1{sub 2}β1γδ) than for the mouse (α1{sub 2}β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.

  4. In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

    Science.gov (United States)

    Malysz, John; Anderson, David J; Grønlien, Jens H; Ji, Jianguo; Bunnelle, William H; Håkerud, Monika; Thorin-Hagene, Kirten; Ween, Hilde; Helfrich, Rosalind; Hu, Min; Gubbins, Earl; Gopalakrishnan, Sujatha; Puttfarcken, Pamela S; Briggs, Clark A; Li, Jinhe; Meyer, Michael D; Dyhring, Tino; Ahring, Philip K; Nielsen, Elsebet Ø; Peters, Dan; Timmermann, Daniel B; Gopalakrishnan, Murali

    2010-09-01

    Enhancement of alpha7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimer's disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective alpha7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to alpha7 nAChRs [rat or human cortex, [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), K(i) = 0.2-0.6 nM or [(3)H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-alpha7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman alpha3beta4, chimeric (alpha6/alpha3)beta4, or 5-HT(3A) receptors, and weak or negligible Ca(2+) responses in human neuroblastoma IMR-32 cells (alpha3* function) and human alpha4beta2 and alpha4beta4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat alpha7 nAChR current responses in oocytes (EC(50), 50-90 nM total charge, approximately 80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked alpha7-like currents, which were inhibited by the alpha7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an alpha7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive alpha7 nAChR-mediated Ca(2+) signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat

  5. The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels

    Directory of Open Access Journals (Sweden)

    Lansdell Stuart J

    2012-06-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3 and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit. Results A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric ‘triplet’ nAChR (Dα5 + Dα6 + Dα7 with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7 with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in

  6. Synergistic effect of sevoflurane and isoflurane on inhibition of the adult-type muscle nicotinic acetylcholine receptor by rocuronium.

    Science.gov (United States)

    Liu, Li; Li, Wei; Wei, Ke; Cao, Jun; Luo, Jie; Wang, Bin; Min, Su

    2013-06-01

    Inhaled anesthetics increase the incidence of postoperative residual neuromuscular blockade, and the mechanism is still unclear. We have investigated the synergistic effect of low-concentration inhaled anesthetics and rocuronium on inhibition of the inward current of the adult-type muscle nicotinic acetylcholine receptor (ε-nAChR). Adult-type mouse muscle ε-nAChR was expressed in HEK293 cells by liposome transfection. The inward current of the ε-nAChR was activated by use of 10 μmol/L acetylcholine alone or in combination with different concentrations of sevoflurane, isoflurane, or rocuronium. The concentration-response curves of five cells were constructed, and the data yielded the 5, 25, and 50 % inhibitory concentrations (IC5, IC25, and IC50, respectively) for single-drug application. Subsequently, the functional channels were perfused by adding 0.5 IC5 of either sevoflurane or isoflurane (aqueous concentrations 140 and 100 μmol/L, respectively) to the solution, followed by addition of IC5, IC25, or IC50 rocuronium. The amount of inhibition was calculated to quantify their synergistic effect. The inhibitory effect of rocuronium was enhanced by sevoflurane or isoflurane in a concentration-dependent manner. Sevoflurane or isoflurane (0.5 IC5) with rocuronium at IC5, IC25, and IC50 synergistically inhibited the current amplitude of adult-type muscle ε-nAChR. When the IC5 of rocuronium was used, isoflurane had a stronger synergistic effect than sevoflurane (p rocuronium was applied at higher concentrations (IC25 and IC50), sevoflurane had an effect similar to that of isoflurane. For both inhaled anesthetics, the synergistic effect was more intense for rocuronium at IC5 than for rocuronium at IC25 or IC50. Residual-concentration sevoflurane or isoflurane has a strong synergistic effect with rocuronium at clinically relevant residual concentrations. A lower rocuronium concentration resulted in a stronger synergistic effect.

  7. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    International Nuclear Information System (INIS)

    Grandič, Marjana; Aráoz, Romulo; Molgó, Jordi; Turk, Tom; Sepčić, Kristina; Benoit, Evelyne; Frangež, Robert

    2012-01-01

    APS12-2, a non-competitive acetylcholinesterase inhibitor, is one of the synthetic analogs of polymeric alkylpyridinium salts (poly-APS) isolated from the marine sponge Reniera sarai. In the present work the effects of APS12-2 were studied on isolated mouse phrenic nerve–hemidiaphragm muscle preparations, using twitch tension measurements and electrophysiological recordings. APS12-2 in a concentration-dependent manner blocked nerve-evoked isometric muscle contraction (IC 50 = 0.74 μM), without affecting directly-elicited twitch tension up to 2.72 μM. The compound (0.007–3.40 μM) decreased the amplitude of miniature endplate potentials until a complete block by concentrations higher than 0.68 μM, without affecting their frequency. Full size endplate potentials, recorded after blocking voltage-gated muscle sodium channels, were inhibited by APS12-2 in a concentration-dependent manner (IC 50 = 0.36 μM) without significant change in the resting membrane potential of the muscle fibers up to 3.40 μM. The compound also blocked acetylcholine-evoked inward currents in Xenopus oocytes in which Torpedo (α1 2 β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC 50 = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α1 2 β1γδ) than for the mouse (α1 2 β1γε) nAChR. Our data show for the first time that APS12-2 blocks neuromuscular transmission by a non-depolarizing mechanism through an action on postsynaptic nAChRs of the skeletal neuromuscular junction. -- Highlights: ► APS12-2 produces concentration-dependent inhibition of nerve-evoked muscle contraction in vitro. ► APS12-2 blocks MEPPs and EPPs at the neuromuscular junction. APS12-2 blocks ACh-activated current in Xenopus oocytes incorporated with Torpedo nAChRs.

  8. Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183)

    International Nuclear Information System (INIS)

    Marien, M.R.; Parsons, S.M.; Altar, C.A.

    1987-01-01

    2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that [ 3 H]AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for [ 3 H]AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound [ 3 H]AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that [ 3 H]AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable [ 3 H]AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, [ 3 H]AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that [ 3 H]AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals

  9. Effect of elevated potassium ion concentrations on electrically evoked release of (/sup 3/H)acetylcholine in slices of rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Szerb, J C; Hadhazy, P; Dudar, J D [Dalhousie Univ., Halifax, Nova Scotia (Canada). Dept. of Physiology and Biophysics

    1978-01-01

    To establish the effect of raising the concentration of extracellular potassium ions on axonal conduction and transmitter release in a mammalian central pathway, the septohippocampal cholinergic tract, the rate of (/sup 3/H) acetylcholine release evoked by electrical stimulation was measured in rat hippocampal slices superfused with Krebs' solution containing 3-15 mM K/sup +/. The evoked release of (/sup 3/H) acetylcholine was abolished by the presence of tetrodotoxin or by the omission of Ca/sup 2 +/ in the superfusion medium, indicating that it originated from terminals depolarized by conducted action potentials. Potassium concentrations between 3 and 8 mM had no effect but 10-15 mM K/sup +/ reduced the rate of evoked release and decreased the size of the releasable pool of (/sup 3/H) acetylcholine. Decreasing the sodium content of the Krebs' solution to 97 mM or less had effects similar to those of elevated (K/sup +/). Elevated K/sup +/ (10-15 mM) reversibly reduced the size of compound action potentials in the fimbria and the alveus. The results suggest that extracellular potassium concentrations occurring under physiological conditions do not affect axonal conduction and transmitter release but that both are reduced in pathological states when extracellular (K/sup +/) above 8 mM occur.

  10. Repeated administration of almonds increases brain acetylcholine levels and enhances memory function in healthy rats while attenuates memory deficits in animal model of amnesia.

    Science.gov (United States)

    Batool, Zehra; Sadir, Sadia; Liaquat, Laraib; Tabassum, Saiqa; Madiha, Syeda; Rafiq, Sahar; Tariq, Sumayya; Batool, Tuba Sharf; Saleem, Sadia; Naqvi, Fizza; Perveen, Tahira; Haider, Saida

    2016-01-01

    Dietary nutrients may play a vital role in protecting the brain from age-related memory dysfunction and neurodegenerative diseases. Tree nuts including almonds have shown potential to combat age-associated brain dysfunction. These nuts are an important source of essential nutrients, such as tocopherol, folate, mono- and poly-unsaturated fatty acids, and polyphenols. These components have shown promise as possible dietary supplements to prevent or delay the onset of age-associated cognitive dysfunction. This study investigated possible protective potential of almond against scopolamine induced amnesia in rats. The present study also investigated a role of acetylcholine in almond induced memory enhancement. Rats in test group were orally administrated with almond suspension (400 mg/kg/day) for four weeks. Both control and almond-treated rats were then divided into saline and scopolamine injected groups. Rats in the scopolamine group were injected with scopolamine (0.5 mg/kg) five minutes before the start of each memory test. Memory was assessed by elevated plus maze (EPM), Morris water maze (MWM) and novel object recognition (NOR) task. Cholinergic function was determined in terms of hippocampal and frontal cortical acetylcholine content and acetylcholinesterase activity. Results of the present study suggest that almond administration for 28 days significantly improved memory retention. This memory enhancing effect of almond was also observed in scopolamine induced amnesia model. Present study also suggests a role of acetylcholine in the attenuation of scopolamine induced amnesia by almond. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice

    International Nuclear Information System (INIS)

    Watson, M.; Ming, X.; McArdle, J.J.

    1989-01-01

    Differences in voluntary ethanol consumption have been noted in various inbred strains of mice and pharmacogenetic approaches have been used to study the mechanisms of action of many drugs such as ethanol. Long-sleep (LS) and short-sleep (SS) mice, selectively bred for differences in ethanol induced narcosis, provide a method by which a relationship between the differential responsiveness of these geno-types and muscarinic acetylcholine receptors (mAChR) may be evaluated. Sleep times after injection of 3ml ethanol/kg (i.p.) verified the higher sensitivity of LS vs. SS. Mean body weights of LS (26.5g) vs. SS (22g) were also significantly (p 3 H](-) quinuclidinylbenzilate ([ 3 H](-)QNB), a specific but nonsubtype selective mAChR antagonist, [ 3 H]pirenzepine ([ 3 H]PZ), a specific M1 mAChR antagonist and [ 3 H]11-2-[[2-[(diethylamino) methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, ([ 3 H]AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K d ) and density (B max ) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) [ 3 H](-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity

  12. Interactions of p-Nitrobenzene Diazonium Fluoroborate and Analogs with the Active Sites of Acetylcholine-Receptor and -Esterase*

    Science.gov (United States)

    Mautner, Henry G.; Bartels, Eva

    1970-01-01

    p-Nitrobenzene diazonium fluoroborate (NDF) is a potent inhibitor of the carbamylcholine-induced depolarization of the electroplax and of acetylcholinesterase. It probably forms covalent bonds with the acetylcholine-receptor and -esterase at the active site of the proteins. Its inhibitory strength is at least the same as that of trimethylammonium diazonium fluoroborate (TDF). The p-acetoxy analog, with its weaker electron-withdrawing group, is about ten times weaker as an inhibitor than the trimethylammonium or p-nitro analogs, both of which have strong electron-withdrawing groups. After treatment of the electroplax preparation with dithiothreitol, NDF remains an irreversible receptor-inhibitor, while TDF becomes a potent reversible receptor-activator. TDF is self-inhibitory: applied before reduction, it no longer depolarizes. Although the first observations on TDF suggested that the compound labels both proteins by virtue of the steric complementary of its trimethylammonium group to a negative subsite in the proteins, the present study indicates that it is the positively charged diazonium group that reacts with the active sites of the proteins to form a covalent bond with an appropriate amino-acid residue. PMID:5272331

  13. CO2-Induced ATP-Dependent Release of Acetylcholine on the Ventral Surface of the Medulla Oblongata.

    Science.gov (United States)

    Huckstepp, Robert T R; Llaudet, Enrique; Gourine, Alexander V

    2016-01-01

    Complex mechanisms that detect changes in brainstem parenchymal PCO2/[H+] and trigger adaptive changes in lung ventilation are responsible for central respiratory CO2 chemosensitivity. Previous studies of chemosensory signalling pathways suggest that at the level of the ventral surface of the medulla oblongata (VMS), CO2-induced changes in ventilation are (at least in part) mediated by the release and actions of ATP and/or acetylcholine (ACh). Here we performed simultaneous real-time biosensor recordings of CO2-induced ATP and ACh release from the VMS in vivo and in vitro, to test the hypothesis that central respiratory CO2 chemosensory transduction involves simultaneous recruitment of purinergic and cholinergic signalling pathways. In anaesthetised and artificially ventilated rats, an increase in inspired CO2 triggered ACh release on the VMS with a peak amplitude of ~5 μM. Release of ACh was only detected after the onset of CO2-induced activation of the respiratory activity and was markedly reduced (by ~70%) by ATP receptor blockade. In horizontal slices of the VMS, CO2-induced release of ATP was reliably detected, whereas CO2 or bath application of ATP (100 μM) failed to trigger release of ACh. These results suggest that during hypercapnia locally produced ATP induces or potentiates the release of ACh (likely from the medullary projections of distal groups of cholinergic neurones), which may also contribute to the development and/or maintenance of the ventilatory response to CO2.

  14. Synthetic localization of a second toxin-binding region within residues α182-198 of Torpedo acetylcholine receptor

    International Nuclear Information System (INIS)

    Mulac-Jericevic, B.; Atassi, M.Z.

    1986-01-01

    A peptide, corresponding to the region 182-198 (peptide αT182-198) of the α chain of Torpedo californica acetylcholine (AChR) was synthesized, purified and characterized. The binding activities of this peptide to α-bungarotoxin and to cobratoxin were studied and compared to the activities of the previously reported synthetic peptide αT125-147 of Torpedo α chain. Binding studies were performed by quantitative radiometric titrations by studying the binding of 125 I-labelled peptides to toxin adsorbents and the binding of 125 I labelled toxins to peptide adsorbents. The specificity of the binding was confirmed by appropriate inhibition experiments. The results showed unequivocally that the α chain of AChR contains a second toxin binding region which resides within, but may not comprise all of, the residues 182-198. The binding of toxins to one synthetic region is inhibited by the other. Thus, the α chain of AChR contains at least two toxin binding regions which may either be two faces of a larger single binding site or, alternatively, two binding sites that are spatially very close and thus the binding of one synthetic region to the toxin site could sterically obstruct the binding of the second synthetic region

  15. Radioligand imaging of α4β2* nicotinic acetylcholine receptors in Alzheimer’s disease and Parkinson’s disease

    International Nuclear Information System (INIS)

    Meyer, P. M.; Tiepolt, S.; Barthel, H.; Hesse, S.; Sabri, O.

    2014-01-01

    The α4β2 * nicotinic acetylcholine receptors (α4β2 * -nAChR) are highly abundant in the human brain. As neuromodulators they play an important role in cognitive functions such as memory, learning and attention as well as mood and motor function. Post mortem studies suggest that abnormalities of α4β2 * -nAChRs are closely linked to histopathological hallmarks of Alzheimer’s disease (AD), such as amyloid aggregates/oligomers and tangle pathology and of Parkinson’s disease (PD) such as Lewy body pathology and the nigrostriatal dopaminergic deficit. In this review we summarize and discuss nicotinic receptor imaging findings of 2-[18F]FA-85380 PET, [ 11 C]nicotine PET and 5-[ 123 I]IA-85380 SPECT studies investigating α4β2 * -nAChR binding in vivo and their relationship to mental dysfunction in the brain of patients with AD and patients out of the spectrum of Lewy body disorders such as PD and Lewy body dementia (DLB). Furthermore, recent developments of novel α4β2*-nAChR-specific PET radioligands, such as (-)[ 18 F]Flubatine or [ 18 F]AZAN are summarized. We conclude that α4β2*-nAChR-specific PET might become a biomarker for early diagnostics and drug developments in patients with AD, DLB and PD, even at early or prodromal stages.

  16. Nicotinic acetylcholine receptor density in cognitively intact subjects at an early stage of Parkinson’s disease

    Directory of Open Access Journals (Sweden)

    Ioannis Ugo eIsaias

    2014-08-01

    Full Text Available We investigated in vivo brain nicotinic acetylcholine receptor (nAChR distribution in cognitively intact subjects with Parkinson's disease (PD at an early stage of the disease. Fourteen patients and 13 healthy subjects were imaged with single photon emission computed tomography (SPECT and the radiotracer 5-[123I]iodo-3-[2(S-2-azetidinylmethoxy]pyridine ([123I]5IA. Patients were selected according to several criteria, including short duration of motor signs (<7 years and normal scores at an extensive neuropsychological evaluation. In PD patients, nAChR density was significantly higher in the putamen, the insular cortex and the supplementary motor area and lower in the caudate nucleus, the orbitofrontal cortex and the middle temporal gyrus. Disease duration positively correlated with nAChR density in the putamen ipsilateral (ρ=0.56, p<0.05 but not contralateral (ρ=0.49, p=0.07 to the clinically most affected hemibody.We observed, for the first time in vivo, higher nAChR density in brain regions of the motor and limbic basal ganglia circuits of subjects with PD. Our findings support the notion of an up-regulated cholinergic activity at the striatal and possibly cortical level in cognitively intact PD patients at an early stage of disease.

  17. Anticonvulsants Based on the α-Substituted Amide Group Pharmacophore Bind to and Inhibit Function of Neuronal Nicotinic Acetylcholine Receptors.

    Science.gov (United States)

    Krivoshein, Arcadius V

    2016-03-16

    Although the antiepileptic properties of α-substituted lactams, acetamides, and cyclic imides have been known for over 60 years, the mechanism by which they act remains unclear. I report here that these compounds bind to the nicotinic acetylcholine receptor (nAChR) and inhibit its function. Using transient kinetic measurements with functionally active, nondesensitized receptors, I have discovered that (i) α-substituted lactams and cyclic imides are noncompetitive inhibitors of heteromeric subtypes (such as α4β2 and α3β4) of neuronal nAChRs and (ii) the binding affinity of these compounds toward the nAChR correlates with their potency in preventing maximal electroshock (MES)-induced convulsions in mice. Based on the hypothesis that α-substituted amide group is the essential pharmacophore of these drugs, I found and tested a simple compound, 2-phenylbutyramide. This compound indeed inhibits nAChR and shows good anticonvulsant activity in mice. Molecular docking simulations suggest that α-substituted lactams, acetamides, and cyclic imides bind to the same sites on the extracellular domain of the receptor. These new findings indicate that inhibition of brain nAChRs may play an important role in the action of these antiepileptic drugs, a role that has not been previously recognized.

  18. A cog in cognition: how the alpha 7 nicotinic acetylcholine receptor is geared towards improving cognitive deficits.

    Science.gov (United States)

    Leiser, Steven C; Bowlby, Mark R; Comery, Thomas A; Dunlop, John

    2009-06-01

    Cognition, memory, and attention and arousal have been linked to nicotinic acetylcholine receptors (nAChRs). Thus it is not surprising that nAChRs have been strongly implicated as therapeutic targets for treating cognitive deficits in disorders such as schizophrenia and Alzheimer's disease (AD). In particular the alpha7 (alpha7) nAChR has been closely linked with normalization of P50 auditory evoked potential (AEP) gating deficits, and to a lesser extent improvements in pre-pulse inhibition (PPI) of the acoustic startle response. These two brain phenomena can be considered as pre-attentive, occurring while sensory information is being processed, and are important endophenotypes in schizophrenia with deficits likely contributing to the cognitive fragmentation associated with the disease. In addition alpha7 nAChRs have been implicated in attention, in particular under high attentional demand, and in more demanding working memory tasks such as long delays in delayed matching tasks. Efficacy of alpha7 nAChR agonists across a range of cognitive processes ranging from pre-attentive to attentive states and working and recognition memory provides a solid basis for their pro-cognitive effects. This review will focus on the recent work highlighting the role of alpha7 in cognition and cognitive processes.

  19. Neuroimmune Interactions in Schizophrenia: Focus on Vagus Nerve Stimulation and Activation of the Alpha-7 Nicotinic Acetylcholine Receptor

    Directory of Open Access Journals (Sweden)

    Fabiana Maria das Graças Corsi-Zuelli

    2017-05-01

    Full Text Available Schizophrenia is one of the most debilitating mental disorders and is aggravated by the lack of efficacious treatment. Although its etiology is unclear, epidemiological studies indicate that infection and inflammation during development induces behavioral, morphological, neurochemical, and cognitive impairments, increasing the risk of developing schizophrenia. The inflammatory hypothesis of schizophrenia is also supported by clinical studies demonstrating systemic inflammation and microglia activation in schizophrenic patients. Although elucidating the mechanism that induces this inflammatory profile remains a challenge, mounting evidence suggests that neuroimmune interactions may provide therapeutic advantages to control inflammation and hence schizophrenia. Recent studies have indicated that vagus nerve stimulation controls both peripheral and central inflammation via alpha-7 nicotinic acetylcholine receptor (α7nAChR. Other findings have indicated that vagal stimulation and α7nAChR-agonists can provide therapeutic advantages for neuropsychiatric disorders, such as depression and epilepsy. This review analyzes the latest results regarding: (I the immune-to-brain pathogenesis of schizophrenia; (II the regulation of inflammation by the autonomic nervous system in psychiatric disorders; and (III the role of the vagus nerve and α7nAChR in schizophrenia.

  20. Neuroimmune Interactions in Schizophrenia: Focus on Vagus Nerve Stimulation and Activation of the Alpha-7 Nicotinic Acetylcholine Receptor

    Science.gov (United States)

    Corsi-Zuelli, Fabiana Maria das Graças; Brognara, Fernanda; Quirino, Gustavo Fernando da Silva; Hiroki, Carlos Hiroji; Fais, Rafael Sobrano; Del-Ben, Cristina Marta; Ulloa, Luis; Salgado, Helio Cesar; Kanashiro, Alexandre; Loureiro, Camila Marcelino

    2017-01-01

    Schizophrenia is one of the most debilitating mental disorders and is aggravated by the lack of efficacious treatment. Although its etiology is unclear, epidemiological studies indicate that infection and inflammation during development induces behavioral, morphological, neurochemical, and cognitive impairments, increasing the risk of developing schizophrenia. The inflammatory hypothesis of schizophrenia is also supported by clinical studies demonstrating systemic inflammation and microglia activation in schizophrenic patients. Although elucidating the mechanism that induces this inflammatory profile remains a challenge, mounting evidence suggests that neuroimmune interactions may provide therapeutic advantages to control inflammation and hence schizophrenia. Recent studies have indicated that vagus nerve stimulation controls both peripheral and central inflammation via alpha-7 nicotinic acetylcholine receptor (α7nAChR). Other findings have indicated that vagal stimulation and α7nAChR-agonists can provide therapeutic advantages for neuropsychiatric disorders, such as depression and epilepsy. This review analyzes the latest results regarding: (I) the immune-to-brain pathogenesis of schizophrenia; (II) the regulation of inflammation by the autonomic nervous system in psychiatric disorders; and (III) the role of the vagus nerve and α7nAChR in schizophrenia. PMID:28620379

  1. Enhancement of acetylcholine-induced desensitization of guinea-pig ileal longitudinal muscle in Ca2+-free conditions.

    Science.gov (United States)

    Horio, S; Nagare, T; Moritoki, H

    1999-10-01

    1. To determine the role of cellular Ca2+ in desensitization, acetylcholine(ACh)-induced desensitization was studied under Ca2+-free condition in guinea-pig ileal longitudinal muscle. 2. Pretreatment of the tissue with 10(-4) M ACh (desensitizing treatment) in normal Tyrode solution caused desensitization of the responses both to ACh and histamine. The desensitizing treatment performed in Ca2+-free solution enhanced desensitization of the responses to ACh and histamine significantly. 3. The desensitizing treatment with ACh caused suppression of the responses to high K+ (tonic component) and Bay K 8644. The desensitizing treatment performed in Ca2+-free solution potentiated the suppression of the responses to high K+ and Bay K 8644 significantly. 4. ACh-induced desensitization was enhanced significantly in the presence of a protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7, 10(-4) M) to a similar extent as desensitization obtained under Ca2+-free condition, but not in the presence of a non-specific and less potent kinase inhibitor, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride (HA1004, 10(-4) M). 5. These results suggested that voltage-gated Ca2+ channels were involved in ACh-induced desensitization and that intracellular Ca2+, which was increased during the stimulation with ACh, inhibited desensitization through the activation of protein kinase C. This kinase could have activated or protected Ca2+ channels during the desensitization process to reduce desensitization.

  2. Self-assembly of SiO2 nanoparticles for the potentiometric detection of neurotransmitter acetylcholine and its inhibitor.

    Science.gov (United States)

    Arruda, Izabela G; Guimarães, Francisco E G; Ramos, Romildo J; Vieira, Nirton C S

    2014-09-01

    The detection and quantification of neurotransmitter acetylcholine (ACh) are relevant because modifications in the ACh levels constitute a threat to human health. The biological regulator of this neurotransmitter is acetylcholinesterase (AChE), an enzyme that catalyzes the hydrolysis of ACh to choline and acetic acid. However, its activity is inhibited in the presence of organophosphate and carbamate pesticides, compromising the degradation of the neurotransmitter. There has been a growing interest in faster and more sensitive detection systems that include new methods and materials for the determination of the ACh concentration. This paper proposes a potentiometric biosensor for the detection of neurotransmitter ACh and its inhibitors, specifically organophosphate pesticide methamidophos. The biosensor is based on a self-assembled platform formed by poly(allylamine) hydrochloride (PAH) and silicon dioxide nanoparticles (SiO2-Np) that contains the immobilized enzyme AChE. First, the responses of the biosensor were investigated for different concentrations of ACh in buffer solutions. After quantifying ACh, the inhibition of AChE in the presence of methamidophos was determined, enabling the quantification of methamidophos expressed as the percentage of enzyme inhibition. The potential advantages of this biosensor include simplicity in building the electrode, possible production on an industrial scale, limited need for qualified personnel to operate the device and low processing cost.

  3. On-line anti-acetylcholine esterase activity of extracts of oxystelma esculentum, aerva javanica and zanthoxylum armatum

    International Nuclear Information System (INIS)

    Murtaza, S.; Ullah, R. S.; Abbas, A.; Riaz, T.; Ghous, T.; Altaf, Y.; Khan, M.; Ahmed, S.

    2013-01-01

    Alzheimer's disease (AD), a disease of brain, resulting in memory impairment and the loss of thinking. The main reason of Alzheimer's disease is firmly associated with some impairment in cholinergic transmission. This impairment may be improved by diminishing the breakdown of acetylcholine at the synaptic site in the brain by inhibiting acetylcholinesterase (AChE). In this work, extracts of three different plants Oxystelma esculentum (OEM), Aerva javanica (AJM) and Zanthoxylum armatum (ZAA) have been screened for their anti-AchE activity. Results of the study demonstrate that of the studied extracts, ZAA (IC/sub 50/ 55.5 micro g/ml) acquired stronger anti-AChE activity. While OEM with IC/sub 50/ 107.2 micro g/ml showed moderate and ZAE and AJM showed weaker action (IC/sub 50/ 182.5 and 275.2 micro g/ml). Galanthamine was used as a positive control (IC/sub 50/ 1.47 micro g/ml). (author)

  4. pH-Responsive Fluorescence Enhancement in Graphene Oxide-Naphthalimide Nanoconjugates: A Fluorescence Turn-On Sensor for Acetylcholine.

    Science.gov (United States)

    Mangalath, Sreejith; Abraham, Silja; Joseph, Joshy

    2017-08-22

    A pH-sensitive, fluorescence "turn-on" sensor based on a graphene oxide-naphthalimide (GO-NI) nanoconjugate for the detection of acetylcholine (ACh) by monitoring the enzymatic activity of acetylcholinesterase (AChE) in aqueous solution is reported. These nanoconjugates were synthesized by covalently anchoring picolyl-substituted NI derivatives on the GO/reduced GO surface through a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide coupling strategy, and the morphological and photophysical properties were studied in detail. Synergistic effects of π-π interactions between GO and the NI chromophore, and efficient photoinduced electron- and energy-transfer processes, were responsible for the strong quenching of fluorescence of these nanoconjugates, which were perturbed under acidic pH conditions, leading to significant enhancement of fluorescence emission. This nanoconjugate was successfully employed for the efficient sensing of pH changes caused by the enzymatic activity of AChE, thereby demonstrating its utility as a fluorescence turn-on sensor for ACh in the neurophysiological range. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Chronic traumatic stress impairs memory in mice: Potential roles of acetylcholine, neuroinflammation and corticotropin releasing factor expression in the hippocampus.

    Science.gov (United States)

    Bhakta, Ami; Gavini, Kartheek; Yang, Euitaek; Lyman-Henley, Lani; Parameshwaran, Kodeeswaran

    2017-09-29

    Chronic stress in humans can result in multiple adverse psychiatric and neurobiological outcomes, including memory deficits. These adverse outcomes can be more severe if each episode of stress is very traumatic. When compared to acute or short term stress relatively little is known about the effects of chronic traumatic stress on memory and molecular changes in hippocampus, a brain area involved in memory processing. Here we studied the effects of chronic traumatic stress in mice by exposing them to adult Long Evan rats for 28 consecutive days and subsequently analyzing behavioral outcomes and the changes in the hippocampus. Results show that stressed mice developed memory deficits when assayed with radial arm maze tasks. However, chronic traumatic stress did not induce anxiety, locomotor hyperactivity or anhedonia. In the hippocampus of stressed mice interleukin-1β protein expression was increased along with decreased corticotropin releasing hormone (CRH) gene expression. Furthermore, there was a reduction in acetylcholine levels in the hippocampus of stressed mice. There were no changes in brain derived neurotrophic factor (BDNF) or nerve growth factor (NGF) levels in the hippocampus of stressed mice. Gene expression of immediate early genes (Zif268, Arc, C-Fos) as well as glucocorticoid and mineralocorticoid receptors were also not affected by chronic stress. These data demonstrate that chronic traumatic stress followed by a recovery period might lead to development of resilience resulting in the development of selected, most vulnerable behavioral alterations and molecular changes in the hippocampus. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Highly Selective and Sensitive Detection of Acetylcholine Using Receptor-Modified Single-Walled Carbon Nanotube Sensors

    Science.gov (United States)

    Xu, Shihong; Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2015-03-01

    Acetylcholine (ACh) is a neurotransmitter in a human central nervous system and is related to various neural functions such as memory, learning and muscle contractions. Dysfunctional ACh regulations in a brain can induce several neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease and myasthenia gravis. In researching such diseases, it is important to measure the concentration of ACh in the extracellular fluid of the brain. Herein, we developed a highly sensitive and selective ACh sensor based on single-walled carbon nanotube-field effect transistors (swCNT-FETs). In our work, M1 mAChR protein, an ACh receptor, was expressed in E.coli and coated on swCNT-FETs with lipid membranes. Here, the binding of ACh onto the receptors could be detected by monitoring the change of electrical currents in the underlying swCNT-FETs, allowing the real-time detection of ACh at a 100 pM concentration. Furthermore, our sensor could selectively detect ACh from other neurotransmitters. This is the first report of the real-time sensing of ACh utilizing specific binding between the ACh and M1 mAChR, and it may lead to breakthroughs in various biomedical applications such as drug screening and disease diagnosis.

  7. Guidelines for standard preclinical experiments in the mouse model of myasthenia gravis induced by acetylcholine receptor immunization.

    Science.gov (United States)

    Tuzun, Erdem; Berrih-Aknin, Sonia; Brenner, Talma; Kusner, Linda L; Le Panse, Rozen; Yang, Huan; Tzartos, Socrates; Christadoss, Premkumar

    2015-08-01

    Myasthenia gravis (MG) is an autoimmune disorder characterized by generalized muscle weakness due to neuromuscular junction (NMJ) dysfunction brought by acetylcholine receptor (AChR) antibodies in most cases. Although steroids and other immunosuppressants are effectively used for treatment of MG, these medications often cause severe side effects and a complete remission cannot be obtained in many cases. For pre-clinical evaluation of more effective and less toxic treatment methods for MG, the experimental autoimmune myasthenia gravis (EAMG) induced by Torpedo AChR immunization has become one of the standard animal models. Although numerous compounds have been recently proposed for MG mostly by using the active immunization EAMG model, only a few have been proven to be effective in MG patients. The variability in the experimental design, immunization methods and outcome measurements of pre-clinical EAMG studies make it difficult to interpret the published reports and assess the potential for application to MG patients. In an effort to standardize the active immunization EAMG model, we propose standard procedures for animal care conditions, sampling and randomization of mice, experimental design and outcome measures. Utilization of these standard procedures might improve the power of pre-clinical EAMG experiments and increase the chances for identifying promising novel treatment methods that can be effectively translated into clinical trials for MG. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.

    Science.gov (United States)

    Allen, Marcus J; Murphey, R K

    2007-07-01

    The largest central synapse in adult Drosophila is a mixed electro-chemical synapse whose gap junctions require the product of the shaking-B (shak-B) gene. Shak-B(2) mutant flies lack gap junctions at this synapse, which is between the giant fibre (GF) and the tergotrochanteral motor neuron (TTMn), but it still exhibits a long latency response upon GF stimulation. We have targeted the expression of the light chain of tetanus toxin to the GF, to block chemical transmission, in shak-B(2) flies. The long latency response in the tergotrochanteral muscle (TTM) was abolished indicating that the chemical component of the synapse mediates this response. Attenuation of GAL4-mediated labelling by a cha-GAL80 transgene, reveals the GF to be cholinergic. We have used a temperature-sensitive allele of the choline acetyltransferase gene (cha(ts2)) to block cholinergic synapses in adult flies and this also abolished the long latency response in shak-B(2) flies. Taken together the data provide evidence that both components of this mixed synapse are functional and that the chemical neurotransmitter between the GF and the TTMn is acetylcholine. Our findings show that the two components of this synapse can be separated to allow further studies into the mechanisms by which mixed synapses are built and function.

  9. Competitive inhibition of the nondepolarizing muscle relaxant rocuronium on nicotinic acetylcholine receptor channels in the rat superior cervical ganglia.

    Science.gov (United States)

    Zhang, Chengmi; Wang, Zhenmeng; Zhang, Jinmin; Qiu, Haibo; Sun, Yuming; Yang, Liqun; Wu, Feixiang; Zheng, Jijian; Yu, Weifeng

    2014-05-01

    A number of case reports now indicate that rocuronium can induce a number of serious side effects. We hypothesized that these side effects might be mediated by the inhibition of nicotinic acetylcholine receptors (nAChRs) at superior cervical ganglion (SCG) neurons. Conventional patch clamp recordings were used to study the effects of rocuronium on nAChR currents from enzymatically dissociated rat SCG neurons. We found that ACh induced a peak transient inward current in rat SCG neurons. Additionally, rocuronium suppressed the peak ACh-evoked currents in rat SCG neurons in a concentration-dependent and competitive manner, and it increased the extent of desensitization of nAChRs. The inhibitory rate of rocuronium on nAChR currents did not change significantly at membrane potentials between -70 and -20 mV, suggesting that this inhibition was voltage independent. Lastly, rocuronium preapplication enhanced its inhibitory effect, indicating that this drug might prefer to act on the closed state of nAChR channels. In conclusion, rocuronium, at clinically relevant concentrations, directly inhibits nAChRs at the SCG by interacting with both opened and closed states. This inhibition is competitive, dose dependent, and voltage independent. Blockade of synaptic transmission in the sympathetic ganglia by rocuronium might have potentially inhibitory effects on the cardiovascular system.

  10. Requirement of Nicotinic Acetylcholine Receptor Subunit β2 in the Maintenance of Spiral Ganglion Neurons during Aging

    Science.gov (United States)

    Bao, Jianxin; Lei, Debin; Du, Yafei; Ohlemiller, Kevin K.; Beaudet, Arthur L.; Role, Lorna W.

    2008-01-01

    Age-related hearing loss (presbycusis) is a major health concern for the elderly. Loss of spiral ganglion neurons (SGNs), the primary sensory relay of the auditory system, is associated consistently with presbycusis. The causative molecular events responsible for age-related loss of SGNs are unknown. Recent reports directly link age-related neuronal loss in cerebral cortex with the loss of high-affinity nicotine acetylcholine receptors (nAChRs). In cochlea, cholinergic synapses are made by olivocochlear efferent fibers on the outer hair cells that express α9 nAChR subunits and on the peripheral projections of SGNs that express α2, α4 –7, and β2–3 nAChR subunits. A significantly decreased expression of the β2 nAChR subunit in SGNs was found specifically in mice susceptible to presbycusis. Furthermore, mice lacking the β2 nAChR subunit (β2−/−), but not mice lacking the α5 nAChR subunit (α5−/−), have dramatic hearing loss and significant reduction in the number of SGNs. Our findings clearly established a requirement for β2 nAChR subunit in the maintenance of SGNs during aging. PMID:15788760

  11. Does N-acetylcysteine have an effect on acetylcholine-induced contractions and histopathological changes on isolated rat ileum?

    International Nuclear Information System (INIS)

    Koc, E.; Yavuzer, Sema A.; Ocakcioglu, B.; Ergun, A.; Can, B.; Saran, Y.

    2007-01-01

    Objective was to investigate the action of N-acetyllcysteine (NAC) on rat isolated ileal contractility and to determine the effects of NAC on histopathological changes on ileal tissue. The study took place at the Faculty of Medicine, Ankara University, Ankara, Turkey, January 2003. Adult Wistar rats were used in all experiments. Two groups were designed. The experimental group, to which NAC 0.5 g/Kg/day was administered orally by adding to their water for 7 days, and the control group to which only saline was administered. At the end of experimental periods, one cm pieces of terminal ileum segments were removed for testing ileal contractility, and one cm pieces of ileum segments were removed for histopathological experiments. The acetylcholine (ACh)-induced contraction was recorded, and the ileal tissue examined using light and electron microscopic techniques for histopathological changes. The average peak amplitude of Ach-induced contraction recorded in standard tyrode solution of the experimental group was decreased significantly when compared to the control group in standard and calcium-free tyrode solution. On histopathological findings, there were swollen mitochondria with disturbed cristae in ileal muscle. Our data suggest that the NAC in the present experiment decreased the ACh-induced contractility on rat-isolated ileum. (author)

  12. Pyridoxal phosphate as a probe of the cytoplasmic domains of transmembrane proteins: Application to the nicotinic acetylcholine receptor

    International Nuclear Information System (INIS)

    Perez-Ramirez, B.; Martinez-Carrion, M.

    1989-01-01

    A novel procedure has been developed to specifically label the cytoplasmic domains of transmembrane proteins with the aldehyde pyridoxal 5-phosphate (PLP). Torpedo californica acetylcholine receptor (AcChR) vesicles were loaded with [ 3 H]pyridoxine 5-phosphate ([ 3 H]PNP) and pyridoxine-5-phosphate oxidase, followed by intravesicular enzymatic oxidation of [ 3 H]PNP at 37 degree C in the presence of externally added cytochrome c as a scavenger of possible leaking PLP product. The four receptor subunits were labeled whether the reaction was carried out on the internal surface or separately designed to mark the external one. On the other hand, the relative pyridoxylation of the subunits differed in both cases, reflecting differences in accessible lysyl residues in each side of the membrane. Even though there are no large differences in the total lysine content among the subunits and there are two copies of the α-subunit, internal surface labeling by PLP was greatest for the highest molecular weight (δ) subunit, reinforcing the concept that the four receptor subunits are transmembranous and may protrude into the cytoplasmic face in a fashion that is proportional to their subunit molecular weight. Yet, the labeling data do not fit well to any of the models proposed for AcChR subunit folding. The method described can be used for selective labeling of the cytoplasmic domains of transmembrane proteins in sealed membrane vesicles

  13. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    Directory of Open Access Journals (Sweden)

    Torrão A.S.

    1997-01-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are ionotropic receptors comprised of a and ß subunits. These receptors are widely distributed in the central nervous system, and previous studies have revealed specific patterns of localization for some nAChR subunits in the vertebrate brain. In the present study we used immunohistochemical methods and monoclonal antibodies to localize the a2, a3, and a5 nAChR subunits in the chick mesencephalon and diencephalon. We observed a differential distribution of these three subunits in the chick brain, and showed that the somata and neuropil of many central structures contain the a5 nAChR subunit. The a2 and a3 subunits, on the other hand, exhibited a more restricted distribution than a5 and other subunits previously studied, namely a7, a8 and ß2. The patterns of distribution of the different nAChR subunits suggest that neurons in many brain structures may contain several subtypes of nAChRs and that in a few regions one particular subtype may determine the cholinergic nicotinic responses

  14. Selective disruption of acetylcholine synthesis in subsets of motor neurons: a new model of late-onset motor neuron disease.

    Science.gov (United States)

    Lecomte, Marie-José; Bertolus, Chloé; Santamaria, Julie; Bauchet, Anne-Laure; Herbin, Marc; Saurini, Françoise; Misawa, Hidemi; Maisonobe, Thierry; Pradat, Pierre-François; Nosten-Bertrand, Marika; Mallet, Jacques; Berrard, Sylvie

    2014-05-01

    Motor neuron diseases are characterized by the selective chronic dysfunction of a subset of motor neurons and the subsequent impairment of neuromuscular function. To reproduce in the mouse these hallmarks of diseases affecting motor neurons, we generated a mouse line in which ~40% of motor neurons in the spinal cord and the brainstem become unable to sustain neuromuscular transmission. These mice were obtained by conditional knockout of the gene encoding choline acetyltransferase (ChAT), the biosynthetic enzyme for acetylcholine. The mutant mice are viable and spontaneously display abnormal phenotypes that worsen with age including hunched back, reduced lifespan, weight loss, as well as striking deficits in muscle strength and motor function. This slowly progressive neuromuscular dysfunction is accompanied by muscle fiber histopathological features characteristic of neurogenic diseases. Unexpectedly, most changes appeared with a 6-month delay relative to the onset of reduction in ChAT levels, suggesting that compensatory mechanisms preserve muscular function for several months and then are overwhelmed. Deterioration of mouse phenotype after ChAT gene disruption is a specific aging process reminiscent of human pathological situations, particularly among survivors of paralytic poliomyelitis. These mutant mice may represent an invaluable tool to determine the sequence of events that follow the loss of function of a motor neuron subset as the disease progresses, and to evaluate therapeutic strategies. They also offer the opportunity to explore fundamental issues of motor neuron biology. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Synthetic α subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

    International Nuclear Information System (INIS)

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

    1986-01-01

    A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) α subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 μg of peptide (T α 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 α subunit. Peptide H α 125-147 differs from T α 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound 125 I-labelled Hα 125-147 antibody bound Hα 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither Hα 125-147 nor T α 125-147. Rats immunized with H α 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 α 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

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

    Energy Technology Data Exchange (ETDEWEB)

    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. Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-05-05

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  18. Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

    Science.gov (United States)

    Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

    2008-06-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

  19. Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

    Science.gov (United States)

    Hancock, Melissa L.; Canetta, Sarah E.; Role, Lorna W.; Talmage, David A.

    2008-01-01

    Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function. PMID:18458158

  20. Alpha-7 nicotinic acetylcholine receptor agonist treatment in a rat model of Huntington's disease and involvement of heme oxygenase-1

    Directory of Open Access Journals (Sweden)

    Laura Foucault-Fruchard

    2018-01-01

    Full Text Available Neuroinflammation is a common element involved in the pathophysiology of neurodegenerative diseases. We recently reported that repeated alpha-7 nicotinic acetylcholine receptor (α7nAChR activations by a potent agonist such as PHA 543613 in quinolinic acid-injured rats exhibited protective effects on neurons. To further investigate the underlying mechanism, we established rat models of early-stage Huntington's disease by injection of quinolinic acid into the right striatum and then intraperitoneally injected 12 mg/kg PHA 543613 or sterile water, twice a day during 4 days. Western blot assay results showed that the expression of heme oxygenase-1 (HO-1, the key component of the cholinergic anti-inflammatory pathway, in the right striatum of rat models of Huntington's disease subjected to intraperitoneal injection of PHA 543613 for 4 days was significantly increased compared to the control rats receiving intraperitoneal injection of sterile water, and that the increase in HO-1 expression was independent of change in α7nAChR expression. These findings suggest that HO-1 expression is unrelated to α7nAChR density and the increase in HO-1 expression likely contributes to α7nAChR activation-related neuroprotective effect in early-stage Huntington's disease.

  1. Pathways of acetylcholine synthesis, transport and release as targets for treatment of adult-onset cognitive dysfunction.

    Science.gov (United States)

    Amenta, F; Tayebati, S K

    2008-01-01

    Acetylcholine (ACh) is a neurotransmitter widely diffused in central, peripheral, autonomic and enteric nervous system. This paper has reviewed the main mechanisms of ACh synthesis, storage, and release. Presynaptic choline transport supports ACh production and release, and cholinergic terminals express a unique transporter critical for neurotransmitter release. Neurons cannot synthesize choline, which is ultimately derived from the diet and is delivered through the blood stream. ACh released from cholinergic synapses is hydrolyzed by acetylcholinesterase into choline and acetyl coenzyme A and almost 50% of choline derived from ACh hydrolysis is recovered by a high-affinity choline transporter. Parallel with the development of cholinergic hypothesis of geriatric memory dysfunction, cholinergic precursor loading strategy was tried for treating cognitive impairment occurring in Alzheimer's disease. Controlled clinical studies denied clinical usefulness of choline and lecithin (phosphatidylcholine), whereas for other phospholipids involved in choline biosynthetic pathways such as cytidine 5'-diphosphocholine (CDP-choline) or alpha-glyceryl-phosphorylcholine (choline alphoscerate) a modest improvement of cognitive dysfunction in adult-onset dementia disorders is documented. These inconsistencies have probably a metabolic explanation. Free choline administration increases brain choline availability but it does not increase ACh synthesis/or release. Cholinergic precursors to serve for ACh biosynthesis should be incorporate and stored into phospholipids in brain. It is probable that appropriate ACh precursors and other correlated molecules (natural or synthesized) could represent a tool for developing therapeutic strategies by revisiting and updating treatments/supplementations coming out from this therapeutic stalemate.

  2. Diadenosine pentaphosphate affects electrical activity in guinea pig atrium via activation of potassium acetylcholine-dependent inward rectifier.

    Science.gov (United States)

    Abramochkin, Denis V; Karimova, Viktoria M; Filatova, Tatiana S; Kamkin, Andre

    2017-07-01

    Diadenosine pentaphosphate (Ap5A) belongs to the family of diadenosine polyphosphates, endogenously produced compounds that affect vascular tone and cardiac performance when released from platelets. The previous findings indicate that Ap5A shortens action potentials (APs) in rat myocardium via activation of purine P2 receptors. The present study demonstrates alternative mechanism of Ap5A electrophysiological effects found in guinea pig myocardium. Ap5A (10 -4  M) shortens APs in guinea pig working atrial myocardium and slows down pacemaker activity in the sinoatrial node. P1 receptors antagonist DPCPX (10 -7  M) or selective GIRK channels blocker tertiapin (10 -6  M) completely abolished all Ap5A effects, while P2 blocker PPADS (10 -4  M) was ineffective. Patch-clamp experiments revealed potassium inward rectifier current activated by Ap5A in guinea pig atrial myocytes. The current was abolished by DPCPX or tertiapin and therefore was considered as potassium acetylcholine-dependent inward rectifier (I KACh ). Thus, unlike rat, in guinea pig atrium Ap5A produces activation of P1 receptors and subsequent opening of KACh channels leading to negative effects on cardiac electrical activity.

  3. Virtual screening studies of Chinese medicine Coptidis Rhizoma as alpha7 nicotinic acetylcholine receptor agonists for treatment of Alzheimer's disease

    Science.gov (United States)

    Xiang, Li; Xu, Youdong; Zhang, Yan; Meng, Xianli; Wang, Ping

    2015-04-01

    Alzheimer's disease (AD) is an age-related neurodegenerative disease. Extensive in vitro and in vivo experiments have proved that the decreased activity of the cholinergic neuron is responsible for the memory and cognition deterioration. The alpha7 nicotinic acetylcholine receptor (α7-nAChR) is proposed to a drug target of AD, and compounds which acting as α7-nAChR agonists are considered as candidates in AD treatment. Chinese medicine CoptidisRhizoma and its compounds are reported in various anti-AD effects. In this study, virtual screening, docking approaches and hydrogen bond analyses were applied to screen potential α7-nAChR agonists from CoptidisRhizome. The 3D structure of the protein was obtained from PDB database. 87 reported compounds were included in this research and their structures were accessed by NCBI Pubchem. Docking analysis of the compounds was performed using AutoDock 4.2 and AutoDock Vina. The images of the binding modes hydrogen bonds and the hydrophobic interaction were rendered with PyMOL1.5.0.4. and LigPlot+ respectively. Finally, N-tran-feruloyltyramine, isolariciresinol, flavanone, secoisolariciresinol, (+)-lariciresinol and dihydrochalcone, exhibited the lowest docking energy of protein-ligand complex. The results indicate these 6 compounds are potential α7 nAChR agonists, and expected to be effective in AD treatment.

  4. A Multi-Route Model of Nicotine-Cotinine Pharmacokinetics, Pharmacodynamics and Brain Nicotinic Acetylcholine Receptor Binding in Humans

    Energy Technology Data Exchange (ETDEWEB)

    Teeguarden, Justin G.; Housand, Conrad; Smith, Jordan N.; Hinderliter, Paul M.; Gunawan, Rudy; Timchalk, Charles

    2013-02-01

    The pharmacokinetics of nicotine, the pharmacologically active alkaloid in tobacco responsible for addiction, are well characterized in humans. We developed a physiologically based pharmacokinetic/pharmacodynamic model of nicotine pharmacokinetics, brain dosimetry and brain nicotinic acetylcholine receptor (nAChRs) occupancy. A Bayesian framework was applied to optimize model parameters against multiple human data sets. The resulting model was consistent with both calibration and test data sets, but in general underestimated variability. A pharmacodynamic model relating nicotine levels to increases in heart rate as a proxy for the pharmacological effects of nicotine accurately described the nicotine related changes in heart rate and the development and decay of tolerance to nicotine. The PBPK model was utilized to quantitatively capture the combined impact of variation in physiological and metabolic parameters, nicotine availability and smoking compensation on the change in number of cigarettes smoked and toxicant exposure in a population of 10,000 people presented with a reduced toxicant (50%), reduced nicotine (50%) cigarette Across the population, toxicant exposure is reduced in some but not all smokers. Reductions are not in proportion to reductions in toxicant yields, largely due to partial compensation in response to reduced nicotine yields. This framework can be used as a key element of a dosimetry-driven risk assessment strategy for cigarette smoke constituents.

  5. In vivo blockade of acetylcholinesterase increases intraovarian acetylcholine and enhances follicular development and fertility in the rat.

    Science.gov (United States)

    Urra, Javier; Blohberger, Jan; Tiszavari, Michelle; Mayerhofer, Artur; Lara, Hernan E

    2016-07-21

    Growth and differentiation of ovarian follicles are regulated by systemic and local factors, which may include acetylcholine (ACh). Granulosa cells (GCs) of growing follicles and luteal cells produce ACh and in cultured GCs it exerts trophic actions via muscarinic receptors. However, such actions were not studied in vivo. After having established that rat ovarian GCs and luteal cells express the ACh-metabolizing enzyme ACh esterase (AChE), we examined the consequences of local application of an AChE inhibitor, huperzine A (HupA), by osmotic minipump delivery into the ovarian bursa of hemiovariectomized rats. Saline was used in the control group. Local delivery of HupA for 4 weeks increased ovarian ACh content. Estrus cyclicity was not changed indicating a locally restricted range of HupA action. The number of primordial and primary follicles was unaffected, but small secondary follicles significantly increased in the HupA group. Furthermore, a significant increase in the number of corpora lutea suggested increased ovulatory events. In support, as shown upon mating, HupA-treated females had significantly increased implantation sites and more pups. Thus the data are in support of a trophic role of ACh in follicular development and ovulation and point to an important role of ACh in female fertility.

  6. On the appearance of acetylcholine receptors in denervated rat diaphragm, and its dependence on nerve stump length

    International Nuclear Information System (INIS)

    Uchitel, O.; Robbins, N.

    1978-01-01

    Acetylcholine (ACh) sensitivity and extrajunctional receptor distribution of the rat diaphragm were closely monitored during the early period following denervation. Both contracture in response to 10 μg/ml of ACh and extrajunctional binding of [ 125 I]alpha-bungarotoxin ([ 125 I]α-BTX) were first detectable 30 h after cutting the phrenic nerve in the thorax. If the nerve were cut more proximally, leaving a 3.5 cm distal nerve stump, the same level of ACh contracture and [ 125 I]α-BTX binding did not appear until 40 h after operation. This 10-h delay was far longer than the 3-h delay in transmission failure reportedly dependent on stump length. The earliest detectable extrajunctional [ 125 I]α-BTX binding appeared throughout the entire muscle fiber, and was not localized to the endplate region as would be expected if degeneration in the nerve terminal induced new receptors. However, later significant increases in [ 125 I]α-BTX binding at the endplate region could have resulted from such degeneration. All these results are consistent with neurotrophic regulation of muscle ACh receptors, working via a mechanism involving axonal transport. (Auth.)

  7. Changes in the α4β2* nicotinic acetylcholine system during chronic controlled alcohol exposure in nonhuman primates.

    Science.gov (United States)

    Hillmer, Ansel T; Tudorascu, Dana L; Wooten, Dustin W; Lao, Patrick J; Barnhart, Todd E; Ahlers, Elizabeth O; Resch, Leslie M; Larson, Julie A; Converse, Alexander K; Moore, Colleen F; Schneider, Mary L; Christian, Bradley T

    2014-05-01

    The precise nature of modifications to the nicotinic acetylcholine receptor (nAChR) system in response to chronic ethanol exposure is poorly understood. The present work used PET imaging to assay α4β2* nAChR binding levels of eight rhesus monkeys before and during controlled chronic ethanol intake. [(18)F]Nifene PET scans were conducted prior to alcohol exposure, and then again after at least 8 months controlled ethanol exposure, including 6 months at 1.5 g/kg/day following a dose escalation period. Receptor binding levels were quantified with binding potentials (BPND) using the cerebellum as a reference region. Alcohol self-administration was assessed as average daily alcohol intake during a 2 month free drinking period immediately following controlled alcohol. Significant decreases in α4β2* nAChR binding were observed in both frontal and insular cortex in response to chronic ethanol exposure. During chronic alcohol exposure, BPND in the lateral geniculate region correlated positively with the amount of alcohol consumed during free drinking. The observed decreases in nAChR availability following chronic alcohol consumption suggest alterations to this receptor system in response to repeated alcohol administration, making this an important target for further study in alcohol abuse and alcohol and nicotine codependence. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. In vitro study of acetylcholine and histamine induced contractions in colon and rectum of adult and neonate rats.

    Science.gov (United States)

    Singh, Shuchita; Mandal, Maloy B

    2013-01-01

    Contractile mechanisms of different parts of the gut in adult and neonate may not be identical due to developmental processes. The present study was undertaken to investigate acetylcholine (ACh) and histamine induced contractile responses of colon and rectum in adult and neonatal albino rats. Contractile responses were recorded from isolated in vitro preparations. The dose-response curve for ACh (0.001-100 microM) revealed dose dependent increase in contractile responses. A significantly (P pheniramine (100 microM) in adult rectum. This potentiating response of pheniramine was absent in neonate rectum. Such effect was also not seen in colon of both adult and neonate. The present investigation indicates that the contractile responses induced by ACh are similar in both adult and neonate, excepting that the blocking effect of atropine in colon was more pronounced in adult as compared to neonate. Further, the results also indicated different mechanism of histamine action in adults and neonates as evidenced by the significant enhancement of contractions by pheniramine only in adult rectum. Therefore, the present results indicate the existence of a different cholinergic and histaminergic activity in adult and neonate as well as in rectal and colonic tissue.

  9. Noradrenaline and acetylcholine responsiveness of glucose-monitoring and glucose-insensitive neurons in the mediodorsal prefrontal cortex.

    Science.gov (United States)

    Nagy, Bernadett; Szabó, István; Csetényi, Bettina; Hormay, Edina; Papp, Szilárd; Keresztes, Dóra; Karádi, Zoltán

    2014-01-16

    The mediodorsal prefrontal cortex (mdPFC), as part of the forebrain glucose-monitoring (GM) system, plays important role in several regulatory processes to control the internal state of the organism and to initiate behavioral outputs accordingly. Little is known, however, about the neurochemical sensitivity of neurons located in this area. Substantial evidence indicates that the locus ceruleus - noradrenaline (NA) projection system and the nucleus basalis magnocellularis - cholinergic projection system regulate behavioral state and state dependent processing of sensory information, various cognitive functions already associated with the mdPFC. The main goal of the present study was to examine noradrenergic and cholinergic responsiveness of glucose-monitoring and glucose-insensitive (GIS) neurons in the mediodorsal prefrontal cortex. One fifth of the neurons tested changed in firing rate to microelectrophoretically applied NA. Responsiveness of the GM cells to this catecholamine proved to be significantly higher than that of the GIS units. Microiontophoretic application of acetylcholine (Ach) resulted in activity changes (predominantly facilitation) of more than 40% of the mdPFC neurons. Proportion of Ach sensitive units among the GM and the GIS neurons was found to be similar. The glucose-monitoring neurons of the mdPFC and their distinct NA and remarkable Ach sensitivity are suggested to be of particular significance in prefrontal control of adaptive behaviors. © 2013 Published by Elsevier B.V.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

    Directory of Open Access Journals (Sweden)

    Simone eBrusco

    2015-09-01

    Full Text Available Regulation of the ‘neuronal’ nicotinic acetylcholine receptors (nAChRs is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine-1-(3-pyridyl-1-butanone (NNK and N’-nitrosonornicotine (NNN on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16 % of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.

  12. A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

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    Sulan Luo

    Full Text Available Conotoxins (CTxs selectively target a range of ion channels and receptors, making them widely used tools for probing nervous system function. Conotoxins have been previously grouped into superfamilies according to signal sequence and into families based on their cysteine framework and biological target. Here we describe the cloning and characterization of a new conotoxin, from Conus vexillum, named αB-conotoxin VxXXIVA. The peptide does not belong to any previously described conotoxin superfamily and its arrangement of Cys residues is unique among conopeptides. Moreover, in contrast to previously characterized conopeptide toxins, which are expressed initially as prepropeptide precursors with a signal sequence, a ''pro'' region, and the toxin-encoding region, the precursor sequence of αB-VxXXIVA lacks a ''pro'' region. The predicted 40-residue mature peptide, which contains four Cys, was synthesized in each of the three possible disulfide arrangements. Investigation of the mechanism of action of αB-VxXXIVA revealed that the peptide is a nicotinic acetylcholine receptor (nAChR antagonist with greatest potency against the α9α10 subtype. (1H nuclear magnetic resonance (NMR spectra indicated that all three αB-VxXXIVA isomers were poorly structured in aqueous solution. This was consistent with circular dichroism (CD results which showed that the peptides were unstructured in buffer, but adopted partially helical conformations in aqueous trifluoroethanol (TFE solution. The α9α10 nAChR is an important target for the development of analgesics and cancer chemotherapeutics, and αB-VxXXIVA represents a novel ligand with which to probe the structure and function of this protein.

  13. Differential Modulation of GABAA and NMDA Receptors by an α7-nicotinic Acetylcholine Receptor Agonist in Chronic Glaucoma

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    Xujiao Zhou

    2017-12-01

    Full Text Available Presynaptic modulation of γ-aminobutyric acid (GABA release by an alpha7 nicotinic acetylcholine receptor (α7-nAChR agonist promotes retinal ganglion cell (RGC survival and function, as suggested by a previous study on a chronic glaucomatous model from our laboratory. However, the role of excitatory and inhibitory amino acid receptors and their interaction with α7-nAChR in physiological and glaucomatous events remains unknown. In this study, we investigated GABAA and N-methyl-D-aspartate (NMDA receptor activity in control and glaucomatous retinal slices and the regulation of amino acid receptor expression and function by α7-nAChR. Whole-cell patch-clamp recordings from RGCs revealed that the α7-nAChR specific agonist PNU-282987 enhanced the amplitude of currents elicited by GABA and reduced the amplitude of currents elicited by NMDA. The positive modulation of GABAA receptor and the negative modulation of NMDA receptor (NMDAR by PNU-282987-evoked were prevented by pre-administration of the α7-nAChR antagonist methyllycaconitine (MLA. The frequency and the amplitude of glutamate receptor-mediated miniature glutamatergic excitatory postsynaptic currents (mEPSCs were not significantly different between the control and glaucomatous RGCs. Additionally, PNU-282987-treated slices showed no alteration in the frequency or amplitude of mEPSCs relative to control RGCs. Moreover, we showed that expression of the α1 subunit of the GABAA receptor was downregulated and the expression of the NMDAR NR2B subunit was upregulated by intraocular pressure (IOP elevation, and the changes of high IOP were blocked by PNU-282987. In conclusion, retina GABAA and NMDARs are modulated positively and negatively, respectively, by activation of α7-nAChR in in vivo chronic glaucomatous models.

  14. A7DB: a relational database for mutational, physiological and pharmacological data related to the α7 nicotinic acetylcholine receptor

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    Sansom Mark SP

    2005-01-01

    Full Text Available Abstract Background Nicotinic acetylcholine receptors (nAChRs are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been α7. This subunit can form functional homomeric pentamers (α75, which can make interpretation of physiological and structural data much simpler. The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner. Description A7DB http://www.lgics.org/a7db/ is a new relational database of manually curated experimental physiological data associated with the α7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the α7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1 a simple text query 2 an incremental query builder 3 an interactive query builder and 4 a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations. Conclusions A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the α7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families.

  15. Biodistribution and radiation dosimetry of the α7 nicotinic acetylcholine receptor ligand [11C]CHIBA-1001 in humans

    International Nuclear Information System (INIS)

    Sakata, Muneyuki; Wu, Jin; Toyohara, Jun; Oda, Keiichi; Ishikawa, Masatomo; Ishii, Kenji; Hashimoto, Kenji; Ishiwata, Kiichi

    2011-01-01

    Introduction: 4-[ 11 C]Methylphenyl 2,4-diazabicyclo[3.2.2]nonane-2-carboxylate ([ 11 C]CHIBA-1001) is a newly developed positron emission tomography (PET) ligand for mapping α 7 nicotinic acetylcholine receptors. We investigated whole-body biodistribution and radiation dosimetry of [ 11 C]CHIBA-1001 in humans and compared the results with those obtained in mice. Methods: Dynamic whole-body PET was carried out for three human subjects after administering a bolus injection of [ 11 C]CHIBA-1001. Emission scans were collected in two-dimensional mode over five bed positions. Regions of interest were placed over 12 organs. Radiation dosimetry was estimated from the residence times of these source organs using the OLINDA program. Biodistribution data from mice were also used for the prediction of radiation dosimetry in humans, and results with and those without accommodation of different proportions of organ-to-total-body mass were compared with the results from the human PET study. Results: In humans, the highest accumulation was observed in the liver, whereas in mice, the highest accumulation was observed in the urinary bladder. The estimated effective dose from the human PET study was 6.9 μSv/MBq, and that from mice was much underestimated. Conclusion: Effective dose estimates for [ 11 C]CHIBA-1001 were compatible with those associated with other common nuclear medicine tests. Absorption doses among several organs were considerably different between the human and mouse studies. Human dosimetry studies for the investigation of radiation safety are desirable as one of the first clinical trials of new PET probes before their application in subsequent clinical investigations.

  16. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    International Nuclear Information System (INIS)

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M.

    2005-01-01

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2 ) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3 H]-quinuclidinyl benzilate ([ 3 H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B max ) and ligand affinity (K d ). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants (K i ). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3 H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies

  17. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells.

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    Elgueta, Claudio; Vielma, Alex H; Palacios, Adrian G; Schmachtenberg, Oliver

    2015-01-01

    Acetylcholine (ACh) is a major retinal neurotransmitter that modulates visual processing through a large repertoire of cholinergic receptors expressed on different retinal cell types. ACh is released from starburst amacrine cells (SACs) under scotopic conditions, but its effects on cells of the rod pathway have not been investigated. Using whole-cell patch clamp recordings in slices of rat retina, we found that ACh application triggers GABA release onto rod bipolar (RB) cells. GABA was released from A17 amacrine cells and activated postsynaptic GABAA and GABAC receptors in RB cells. The sensitivity of ACh-induced currents to nicotinic ACh receptor (nAChR) antagonists (TMPH ~ mecamylamine > erysodine > DhβE > MLA) together with the differential potency of specific agonists to mimic ACh responses (cytisine > RJR2403 ~ choline), suggest that A17 cells express heteromeric nAChRs containing the β4 subunit. Activation of nAChRs induced GABA release after Ca(2+) accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca(2+) stores. Inhibition of acetylcholinesterase depolarized A17 cells and increased spontaneous inhibitory postsynaptic currents in RB cells, indicating that endogenous ACh enhances GABAergic inhibition of RB cells. Moreover, injection of neostigmine or cytisine reduced the b-wave of the scotopic flash electroretinogram (ERG), suggesting that cholinergic modulation of GABA release controls RB cell activity in vivo. These results describe a novel regulatory mechanism of RB cell inhibition and complement our understanding of the neuromodulatory control of retinal signal processing.

  18. Prior nicotine self-administration attenuates subsequent dopaminergic deficits of methamphetamine in rats: role of nicotinic acetylcholine receptors.

    Science.gov (United States)

    Baladi, Michelle G; Nielsen, Shannon M; McIntosh, J Michael; Hanson, Glen R; Fleckenstein, Annette E

    2016-08-01

    Preclinical studies have demonstrated that oral nicotine exposure attenuates long-term dopaminergic damage induced by toxins, including repeated, high doses of methamphetamine. It is suggested that alterations in nicotinic acetylcholine receptor (nAChR) expression, including α4β2* and α6β2* subtypes, likely contribute to this protection. The current study extended these findings by investigating whether nicotine self-administration in male, Sprague-Dawley rats (a) attenuates short-term dopaminergic damage induced by methamphetamine and (b) causes alterations in levels of α4β2* and α6β2* nAChR subtypes. The findings indicate that nicotine self-administration (0.032 mg/kg/infusion for 14 days) per se did not alter α4β2* and α6β2* nAChR expression or dopamine transporter (DAT) expression and function. Interestingly, prior nicotine self-administration attenuated methamphetamine-induced decreases in DAT function when assessed 24 h, but not 1 h, after methamphetamine treatment (4×7.5 mg/kg/injection). The ability of nicotine to attenuate the effects of methamphetamine on DAT function corresponded with increases in α4β2*, but not α6β2*, nAChR binding density. Understanding the role of nAChRs in methamphetamine-induced damage has the potential to elucidate mechanisms underlying the etiology of disorders involving dopaminergic dysfunction, as well as to highlight potential new therapeutic strategies for prevention or reduction of dopaminergic neurodegeneration.

  19. Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion.

    Science.gov (United States)

    Thany, Steeve H

    2009-11-01

    Clothianidin is new neonicotinoid insecticide acting selectively on insect nicotinic acetylcholine receptors (nAChRs). Its effects on nAChRs expressed on cercal afferent/giant interneuron synapses and DUM neurons have been studied using mannitol-gap and whole-cell patch-clamp techniques, respectively. Bath-application of clothianidin-induced dose-dependent depolarizations of cockroach cercal afferent/giant interneuron synapses which were not reversed after wash-out suggesting a strong desensitization of postsynaptic interneurons at the 6th abdominal ganglion (A6). Clothinidin activity on the nerve preparation was characterized by an increased firing rate of action potentials which then ceased when the depolarization reached a peak. Clothianidin responses were insensitive to all muscarinic antagonists tested but were blocked by co-application of specific nicotinic antagonists methyllicaconitine, alpha-bungarotoxin and d-tubocurarine. In a second round of experiment, clothianidin actions were tested on DUM neurons isolated from the A6. There was a strong desensitization of nAChRs which was not affected by muscarinic antagonists, pirenzepine and atropine, but was reduced with nicotinic antagonist alpha-bungarotoxin. In addition, clothianidin-induced currents were completely blocked by methyllicaconitine suggesting that (1) clothianidin acted as a specific agonist of nAChR subtypes and (2) a small proportion of receptors blocked by MLA was insensitive to alpha-bungarotoxin. Moreover, because clothianidin currents were blocked by d-tubocurarine and mecamylamine, we provided that clothianidin was an agonist of both nAChRs: imidacloprid-sensitive nAChR1 and -insensitive nAChR2 subtypes.

  20. α6β2* and α4β2* Nicotinic Acetylcholine Receptors As Drug Targets for Parkinson's Disease

    Science.gov (United States)

    Wonnacott, Susan

    2011-01-01

    Parkinson's disease is a debilitating movement disorder characterized by a generalized dysfunction of the nervous system, with a particularly prominent decline in the nigrostriatal dopaminergic pathway. Although there is currently no cure, drugs targeting the dopaminergic system provide major symptomatic relief. As well, agents directed to other neurotransmitter systems are of therapeutic benefit. Such drugs may act by directly improving functional deficits in these other systems, or they may restore aberrant motor activity that arises as a result of a dopaminergic imbalance. Recent research attention has focused on a role for drugs targeting the nicotinic cholinergic systems. The rationale for such work stems from basic research findings that there is an extensive overlap in the organization and function of the nicotinic cholinergic and dopaminergic systems in the basal ganglia. In addition, nicotinic acetylcholine receptor (nAChR) drugs could have clinical potential for Parkinson's disease. Evidence for this proposition stems from studies with experimental animal models showing that nicotine protects against neurotoxin-induced nigrostriatal damage and improves motor complications associated with l-DOPA, the “gold standard” for Parkinson's disease treatment. Nicotine interacts with multiple central nervous system receptors to generate therapeutic responses but also produces side effects. It is important therefore to identify the nAChR subtypes most beneficial for treating Parkinson's disease. Here we review nAChRs with particular emphasis on the subtypes that contribute to basal ganglia function. Accumulating evidence suggests that drugs targeting α6β2* and α4β2* nAChR may prove useful in the management of Parkinson's disease. PMID:21969327

  1. Partial agonism at the α7 nicotinic acetylcholine receptor improves attention, impulsive action and vigilance in low attentive rats.

    Science.gov (United States)

    Hayward, Andrew; Adamson, Lisa; Neill, Joanna C

    2017-04-01

    Inattention is a disabling symptom in conditions such as schizophrenia and attention deficit/hyperactivity disorder. Nicotine can improve attention and vigilance, but is unsuitable for clinical use due to abuse liability. Genetic knockout of the α7 nicotinic acetylcholine receptor (nAChR) induces attention deficits therefore selective agonism may improve attention, without the abuse liability associated with nicotine. The α7 nAChR partial agonist encenicline (formerly EVP-6124) enhances memory in rodents and humans. Here we investigate, for the first time, efficacy of encenicline to improve attention and vigilance in animals behaviourally grouped for low attentive traits in the 5 choice-continuous performance task (5C-CPT). Female Lister Hooded rats were trained to perform the 5C-CPT with a variable stimulus duration (SD). Animals were then grouped based on performance into upper and lower quartiles of d' (vigilance) and accuracy (selective attention), producing high-attentive (HA) and low-attentive (LA) groups. LA animals showed an increase in selective attention and vigilance at 0.3mg/kg encenicline, a reduction in impulsive action (probability of false alarms) and increase in vigilance following 1mg/kg at 0.75sSD. At 1mg/kg, HA animals had reduced selective attention at 0.75sSD and reduced vigilance at 0.75 and 1.25sSD. Improvement of attention, vigilance and impulsive action in LA animals demonstrates that encenicline has pro-attentive properties dependent on baseline levels of performance. Our work suggests that α7 nAChR partial agonism may improve attention particularly in conditions with low attention. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  2. Amino acid and acetylcholine chemistry in the central auditory system of young, middle-aged and old rats.

    Science.gov (United States)

    Godfrey, Donald A; Chen, Kejian; O'Toole, Thomas R; Mustapha, Abdurrahman I A A

    2017-07-01

    Older adults generally experience difficulties with hearing. Age-related changes in the chemistry of central auditory regions, especially the chemistry underlying synaptic transmission between neurons, may be of particular relevance for hearing changes. In this study, we used quantitative microchemical methods to map concentrations of amino acids, including the major neurotransmitters of the brain, in all the major central auditory structures of young (6 months), middle-aged (22 months), and old (33 months old) Fischer 344 x Brown Norway rats. In addition, some amino acid measurements were made for vestibular nuclei, and activities of choline acetyltransferase, the enzyme for acetylcholine synthesis, were mapped in the superior olive and auditory cortex. In old, as compared to young, rats, glutamate concentrations were lower throughout central auditory regions. Aspartate and glycine concentrations were significantly lower in many and GABA and taurine concentrations in some cochlear nucleus and superior olive regions. Glutamine concentrations and choline acetyltransferase activities were higher in most auditory cortex layers of old rats as compared to young. Where there were differences between young and old rats, amino acid concentrations in middle-aged rats often lay between those in young and old rats, suggesting gradual changes during adult life. The results suggest that hearing deficits in older adults may relate to decreases in excitatory (glutamate) as well as inhibitory (glycine and GABA) neurotransmitter amino acid functions. Chemical changes measured in aged rats often differed from changes measured after manipulations that directly damage the cochlea, suggesting that chemical changes during aging may not all be secondary to cochlear damage. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Regional relation between skin blood flow and sweating to passive heating and local administration of acetylcholine in young, healthy humans.

    Science.gov (United States)

    Smith, Caroline J; Kenney, W Larry; Alexander, Lacy M

    2013-04-01

    Regional variation in sweating over the human body is widely recognized yet variation in vasomotor responses and mechanisms causing this variation remain unclear. This study aimed to explore the relation between regional sweating rates (RSR) and skin blood flow (SkBF) responses to thermal and pharmacological stimuli in young, healthy subjects. In nine subjects (23 ± 3 yr), intradermal microdialysis (MD) probes were inserted into the ventral forearm, abdomen, thigh, and lower back and perfused with lactated Ringer solution. RSR over each MD membrane were measured using ventilated capsules with a laser Doppler probe housed in each capsule for measurement of red cell flux (laser Doppler flux, LDF) as an index of SkBF. Subjects completed a whole body heating protocol to 1°C rise in oral temperature and an acetylcholine dose response (ACh 1 × 10(-7)-0.1 M; mean skin temperature 34°C). Maximal LDF were obtained at the end of both protocols (50 mM sodium nitroprusside).During heating RSR varied among sites (P back versus other sites (P back: r = 0.86 ± 0.04) but not latter stages of heating. No differences in RSR (P = 0.160) or SkBF (LDF, P = 0.841) were observed between sites during ACh perfusion. Taken together, these data suggest that increases in SkBF are necessary to initiate and increase sweating, but further rises in RSR are not fully dependent on SkBF in a dose-response manner. Furthermore, RSR cannot be explained by cholinergic sensitivity or variation in SkBF.

  4. Facilitation of acetylcholine release in rat frontal cortex by indeloxazine hydrochloride: involvement of endogenous serotonin and 5-HT4 receptors.

    Science.gov (United States)

    Yamaguchi, T; Suzuki, M; Yamamoto, M

    1997-12-01

    Effects of indeloxazine hydrochloride, an inhibitor of serotonin (5-HT) and norepinephrine (NE) reuptake with a facilitatory effect on 5-HT release, on acetylcholine (ACh) output in frontal cortex of conscious rats were characterized using an in vivo microdialysis technique. Systemic administration of indeloxazine (3 and 10 mg/kg, i.p.) increased ACh and 5-HT output in a dose-dependent manner. Depletion of endogenous monoamines by reserpine and of 5-HT by p-chlorophenylalanine, but not that of catecholamines by alpha-methyl-p-tyrosine, significantly attenuated the facilitatory effect of indeloxazine on ACh release. When applied locally by reverse dialysis, indeloxazine (10 and 30 microM) and the selective 5-HT reuptake inhibitor citalopram (10 microM), but not the NE reuptake inhibitor maprotiline (30 microM), increased cortical ACh output. Indeloxazine (10 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT4 receptor antagonists RS23597 (50 microM) and GR113803 (1 microM), while the 5-HT1A antagonist WAY-100135 (100 microM), 5-HT1A/1B/beta-adrenoceptor antagonist (-)propranolol (150 microM), 5-HT2A/2C antagonist ritanserin (10 microM) and 5-HT3 antagonist ondansetron (10 microM) failed to significantly modify this effect. Neither depletion of monoamines nor treatment with serotonergic antagonists significantly changed the basal ACh level, indicating that endogenous monoamines do not tonically activate ACh release. These results suggest that indeloxazine-induced facilitation of ACh release in rat frontal cortex is mediated by endogenous 5-HT and involves at least in part cortical 5-HT4 receptors.

  5. BAY 41-2272 Treatment Improves Acetylcholine-Induced Aortic Relaxation in L-NAME Hypertensive Rats.

    Science.gov (United States)

    Prawez, Shahid; Ahanger, Azad Ahmad; Singh, Thakur Uttam; Mishra, Santosh Kumar; Sarkar, Souvendra Nath; Kumar, Dinesh

    2016-12-01

    Hypertension, an emerging problem of recent era, and many pathophysiological factors are participating to produce the disease. Nitric oxide (NO) is an important constituent to ameliorate hypertensive condition. Inhibition of endogenous NO synthase by L-N G -Nitroarginine methyl ester (L-NAME) was responsible for generating hypertension in rats. BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine), a soluble guanylyl cyclase activator, restricts rise of blood pressure and shows cardioprotective activity. The aim of the present study was to analyze effect of short-term BAY 41-2272 treatment on blood pressure and vascular function. Male Wistar rats were randomly divided into three groups such as control (group-A), hypertensive (group-B), and BAY 41-2272-treated hypertensive (group-C) rats. Normal saline was administered intramuscularly to control rats for last 3 days (days 40, 41, and 42) of total 42 days treatment, whereas rats of group-B and group-C were treated with L-NAME hydrochloride in drinking water at 50 mg/kg body weight daily for 42 days. Also, normal saline and BAY 41-2272 were administered for last 3 days at two different dosages at 1 and 3 mg/kg body weight/day intramuscularly to group-B and group-C rats, respectively. Administration of BAY 41-2272 for 3 days was not sufficient enough to decrease mean arterial pressure of hypertensive rats significantly. BAY at both the treatment dosages significantly ameliorate acetylcholine-induced maximal aortic relaxation compared with BAY-untreated hypertensive rats. Findings of the present study indicate that even shorter period of BAY 41-2272 treatment (3 days) improves vascular relaxation.

  6. Activation of 5-HT2 receptors enhances the release of acetylcholine in the prefrontal cortex and hippocampus of the rat.

    Science.gov (United States)

    Nair, Sunila G; Gudelsky, Gary A

    2004-09-15

    The role of 5-HT2 receptors in the regulation of acetylcholine (ACh) release was examined in the medial prefrontal cortex and dorsal hippocampus using in vivo microdialysis. The 5-HT(2A/2C) agonist +/-1-(2,5-dimethoxy-4-iodophenyl) -2- aminopropane hydrochloride (DOI) (1 and 2 mg/kg, i.p.) significantly increased the extracellular concentration of ACh in both brain regions, and this response was attenuated in rats treated with the 5-HT(2A/2B/2C) antagonist LY-53,857 (3 mg/kg, i.p.). Treatment with LY-53,857 alone did not significantly alter ACh release in either brain region The 5-HT(2C) agonist 6-chloro-2-(1-piperazinyl)-pyrazine) (MK-212) (5 mg/kg, i.p.) significantly enhanced the release of ACh in both the prefrontal cortex and hippocampus, whereas the 5-HT2 agonist mescaline (10 mg/kg, i.p.) produced a 2-fold increase in ACh release only in the prefrontal cortex. Intracortical, but not intrahippocampal, infusion of DOI (100 microM) significantly enhanced the release of ACh, and intracortical infusion of LY-53,857 (100 microM) significantly attenuated this response. These results suggest that the release of ACh in the prefrontal cortex and hippocampus is influenced by 5-HT2 receptor mechanisms. The increase in release of ACh induced by DOI in the prefrontal cortex, but not in the hippocampus, appears to be due to 5-HT2 receptor mechanisms localized within this brain region. Furthermore, it appears that the prefrontal cortex is more sensitive than the dorsal hippocampus to the stimulatory effect of 5-HT2 agonists on ACh release.

  7. Characterization of 5-hydroxytryptamine-induced contraction and acetylcholine-induced relaxation in isolated chicken basilar artery.

    Science.gov (United States)

    Matsumoto, F; Watanabe, Y; Obi, T; Islam, M Z; Yamazaki-Himeno, E; Shiraishi, M; Miyamoto, A

    2012-05-01

    The aim of the present study was to clarify the responsiveness of the chicken basilar artery to 5-hydroxytryptamine (5-HT) and acetylcholine (ACh) and to characterize the related receptor subtypes in vitro. Basilar arteries were obtained from freshly slaughtered broiler chickens. The 5-HT induced concentration-dependent contraction of the arteries. The concentration-response curves for 5-HT were shifted 30-fold to the right by methiothepin (a 5-HT(1) and 5-HT(2) receptor antagonist) and 3-fold to the right by ketanserin (a 5-HT(2) receptor antagonist). In the presence of ketanserin, the concentration-response curve for 5-HT was shifted 10-fold to the right by methiothepin. The pA(2) value for methiothepin was 8.26. The ACh induced concentration-dependent relaxation under conditions of precontraction by 5-HT. The concentration-response curve for ACh was shifted to the right by atropine [a nonselective muscarinic (M) receptor antagonist] and hexahydro-sila-difenidol hydrochloride, a p-fluoroanalog (pFHHSiD, an M(3) receptor antagonist), but not by pirenzepine (an M(1) receptor antagonist) or methoctramine (an M(2) receptor antagonist). The pA(2) value for pFHHSiD was 7.55. Nω-Nitro-l-arginine (a nitric oxide synthase inhibitor) inhibited ACh-induced relaxation by approximately 50%. These results suggest that 5-HT induces contraction via activation of 5-HT(1) and 5-HT(2) receptors and that ACh induces relaxation via activation of the M(3) receptor. The 5-HT(1) receptor might play a dominant role in 5-HT-induced contraction. One of the factors involved in ACh-induced relaxation is probably nitric oxide released from endothelial cells.

  8. Prorocentrolide-A from Cultured Prorocentrum lima Dinoflagellates Collected in Japan Blocks Sub-Types of Nicotinic Acetylcholine Receptors

    Directory of Open Access Journals (Sweden)

    Muriel Amar

    2018-02-01

    Full Text Available Prorocentrolides are members of the cyclic imine phycotoxins family. Their chemical structure includes a 26-membered carbo-macrocycle and a 28-membered macrocyclic lactone arranged around a hexahydroisoquinoline that incorporates the characteristic cyclic imine group. Six prorocentrolides are already known. However, their mode of action remains undetermined. The aim of the present work was to explore whether prorocentrolide-A acts on nicotinic acetylcholine receptors (nAChRs, using competition-binding assays and electrophysiological techniques. Prorocentrolide-A displaced [125I]α-bungarotoxin binding to Torpedo membranes, expressing the muscle-type (α12β1γδ nAChR, and in HEK-293 cells, expressing the chimeric chick neuronal α7-5HT3 nAChR. Functional studies revealed that prorocentrolide-A had no agonist action on nAChRs, but inhibited ACh-induced currents in Xenopus oocytes that had incorporated the muscle-type α12β1γδ nAChR to their membranes, or that expressed the human α7 nAChR, as revealed by voltage-clamp recordings. Molecular docking calculations showed the absence of the characteristic hydrogen bond between the iminium group of prorocentrolide-A and the backbone carbonyl group of Trp147 in the receptor, explaining its weaker affinity as compared to all other cyclic imine toxins. In conclusion, this is the first study to show that prorocentrolide-A acts on both muscle and neuronal nAChRs, but with higher affinity on the muscle-type nAChR.

  9. Inhibition of the nicotinic acetylcholine receptors by cobra venom α-neurotoxins: is there a perspective in lung cancer treatment?

    Directory of Open Access Journals (Sweden)

    Angela Alama

    Full Text Available Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated.We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin and Naja kaouthia (long-chain neurotoxin, α-cobratoxin in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID mouse model system utilizing different treatment schedules and dosages.No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached.In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals.

  10. A nicotinic acetylcholine receptor mutation conferring target-site resistance to imidacloprid in Nilaparvata lugens (brown planthopper).

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    Liu, Zewen; Williamson, Martin S; Lansdell, Stuart J; Denholm, Ian; Han, Zhaojun; Millar, Neil S

    2005-06-14

    Neonicotinoids, such as imidacloprid, are nicotinic acetylcholine receptor (nAChR) agonists with potent insecticidal activity. Since its introduction in the early 1990s, imidacloprid has become one of the most extensively used insecticides for both crop protection and animal health applications. As with other classes of insecticides, resistance to neonicotinoids is a significant threat and has been identified in several pest species, including the brown planthopper, Nilaparvata lugens, a major rice pest in many parts of Asia. In this study, radioligand binding experiments have been conducted with whole-body membranes prepared from imidacloprid-susceptible and imidacloprid-resistant strains of N. lugens. The results reveal a much higher level of [3H]imidacloprid-specific binding to the susceptible strain than to the resistant strain (16.7 +/- 1.0 and 0.34 +/- 0.21 fmol/mg of protein, respectively). With the aim of understanding the molecular basis of imidacloprid resistance, five nAChR subunits (Nlalpha1-Nlalpha4 and Nlbeta1) have been cloned from N. lugens.A comparison of nAChR subunit genes from imidacloprid-sensitive and imidacloprid-resistant populations has identified a single point mutation at a conserved position (Y151S) in two nAChR subunits, Nlalpha1 and Nlalpha3. A strong correlation between the frequency of the Y151S point mutation and the level of resistance to imidacloprid has been demonstrated by allele-specific PCR. By expression of hybrid nAChRs containing N. lugens alpha and rat beta2 subunits, evidence was obtained that demonstrates that mutation Y151S is responsible for a substantial reduction in specific [3H]imidacloprid binding. This study provides direct evidence for the occurrence of target-site resistance to a neonicotinoid insecticide.

  11. Effect of ispronicline, a neuronal nicotinic acetylcholine receptor partial agonist, in subjects with age associated memory impairment (AAMI).

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    Dunbar, Geoffrey C; Inglis, Fraser; Kuchibhatla, Ramana; Sharma, Tonmoy; Tomlinson, Mark; Wamsley, James

    2007-03-01

    Cognitive decline seen in the normal elderly is associated with selective loss of neuronal nicotinic acetylcholine receptors (nAChRs). Nicotine given either by inhalation or transdermally helps cognition, but unacceptable side effects limit its utility. The present study assessed the safety, tolerability and effect on cognition of ispronicline, a highly selective partial agonist at the 4beta2 nAChR, in elderly subjects (n =76) with age associated memory impairment (AAMI). This double-blind, placebo-controlled cross-over study explored ascending oral doses of ispronicline in the range 50-150 mg given as a single morning dose for a period of 3 weeks. Pharmacokinetics (PK) were assessed, as well as cognitive function measured by means of the Cognitive Drug Research (CDR) computerized test battery. Ispronicline had a favourable safety profile and was well tolerated at doses below 150 mg. No effect of clinical importance was seen on biochemistry, haematology, urine analysis, vital signs, electrocardiogram (ECG) or Holter monitoring. The most frequent drug induced adverse event was light-headedness (dizziness). A beneficial effect was seen on cognition across the dose range. This was most marked at 50 mg on factors measuring attention and episodic memory. PK analysis indicated a plasma Cmax range of 5-25/35 ng/ml ispronicline was associated with the most beneficial effect. These early results demonstrate ispronicline was well tolerated and did not display the side effects typical of nicotine. Ispronicline also had a beneficial effect on cognition in subjects with AAMI. This was seen most strongly in a Cmax range that had been predicted from pre-clinical animal studies.

  12. Joint association of nicotinic acetylcholine receptor variants with abdominal obesity in American Indians: the Strong Heart Family Study.

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    Zhu, Yun; Yang, Jingyun; Yeh, Fawn; Cole, Shelley A; Haack, Karin; Lee, Elisa T; Howard, Barbara V; Zhao, Jinying

    2014-01-01

    Cigarette smoke is a strong risk factor for obesity and cardiovascular disease. The effect of genetic variants involved in nicotine metabolism on obesity or body composition has not been well studied. Though many genetic variants have previously been associated with adiposity or body fat distribution, a single variant usually confers a minimal individual risk. The goal of this study is to evaluate the joint association of multiple variants involved in cigarette smoke or nicotine dependence with obesity-related phenotypes in American Indians. To achieve this goal, we genotyped 61 tagSNPs in seven genes encoding nicotine acetylcholine receptors (nAChRs) in 3,665 American Indians participating in the Strong Heart Family Study. Single SNP association with obesity-related traits was tested using family-based association, adjusting for traditional risk factors including smoking. Joint association of all SNPs in the seven nAChRs genes were examined by gene-family analysis based on weighted truncated product method (TPM). Multiple testing was controlled by false discovery rate (FDR). Results demonstrate that multiple SNPs showed weak individual association with one or more measures of obesity, but none survived correction for multiple testing. However, gene-family analysis revealed significant associations with waist circumference (p = 0.0001) and waist-to-hip ratio (p = 0.0001), but not body mass index (p = 0.20) and percent body fat (p = 0.29), indicating that genetic variants are jointly associated with abdominal, but not general, obesity among American Indians. The observed combined genetic effect is independent of cigarette smoking per se. In conclusion, multiple variants in the nAChR gene family are jointly associated with abdominal obesity in American Indians, independent of general obesity and cigarette smoking per se.

  13. Clonal heterogeneity of thymic B cells from early-onset myasthenia gravis patients with antibodies against the acetylcholine receptor.

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    Vrolix, Kathleen; Fraussen, Judith; Losen, Mario; Stevens, Jo; Lazaridis, Konstantinos; Molenaar, Peter C; Somers, Veerle; Bracho, Maria Alma; Le Panse, Rozen; Stinissen, Piet; Berrih-Aknin, Sonia; Maessen, Jos G; Van Garsse, Leen; Buurman, Wim A; Tzartos, Socrates J; De Baets, Marc H; Martinez-Martinez, Pilar

    2014-08-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR-MG) is considered as a prototypic autoimmune disease. The thymus is important in the pathophysiology of the disease since thymus hyperplasia is a characteristic of early-onset AChR-MG and patients often improve after thymectomy. We hypothesized that thymic B cell and antibody repertoires of AChR-MG patients differ intrinsically from those of control individuals. Using immortalization with Epstein-Barr Virus and Toll-like receptor 9 activation, we isolated and characterized monoclonal B cell lines from 5 MG patients and 8 controls. Only 2 of 570 immortalized B cell clones from MG patients produced antibodies against the AChR (both clones were from the same patient), suggesting that AChR-specific B cells are not enriched in the thymus. Surprisingly, many B cell lines from both AChR-MG and control thymus samples displayed reactivity against striated muscle proteins. Striational antibodies were produced by 15% of B cell clones from AChR-MG versus 6% in control thymus. The IgVH gene sequence analysis showed remarkable similarities, concerning VH family gene distribution, mutation frequency and CDR3 composition, between B cells of AChR-MG patients and controls. MG patients showed clear evidence of clonal B cell expansion in contrast to controls. In this latter aspect, MG resembles multiple sclerosis and clinically isolated syndrome, but differs from systemic lupus erythematosus. Our results support an antigen driven immune response in the MG thymus, but the paucity of AChR-specific B cells, in combination with the observed polyclonal expansions suggest a more diverse immune response than expected. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Nicotine signals through muscle-type and neuronal nicotinic acetylcholine receptors in both human bronchial epithelial cells and airway fibroblasts

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    Luketich James D

    2004-12-01

    Full Text Available Abstract Background Non-neuronal cells, including those derived from lung, are reported to express nicotinic acetylcholine receptors (nAChR. We examined nAChR subunit expression in short-term cultures of human airway cells derived from a series of never smokers, ex-smokers, and active smokers. Methods and Results At the mRNA level, human bronchial epithelial (HBE cells and airway fibroblasts expressed a range of nAChR subunits. In multiple cultures of both cell types, mRNA was detected for subunits that constitute functional muscle-type and neuronal-type pentomeric receptors. Two immortalized cell lines derived from HBE cells also expressed muscle-type and neuronal-type nAChR subunits. Airway fibroblasts expressed mRNA for three muscle-type subunits (α1, δ, and ε significantly more often than HBE cells. Immunoblotting of HBE cell and airway fibroblast extracts confirmed that mRNA for many nAChR subunits is translated into detectable levels of protein, and evidence of glycosylation of nAChRs was observed. Some minor differences in nAChR expression were found based on smoking status in fibroblasts or HBE cells. Nicotine triggered calcium influx in the immortalized HBE cell line BEAS2B, which was blocked by α-bungarotoxin and to a lesser extent by hexamethonium. Activation of PKC and MAPK p38, but not MAPK p42/44, was observed in BEAS2B cells exposed to nicotine. In contrast, nicotine could activate p42/44 in airway fibroblasts within five minutes of exposure. Conclusions These results suggest that muscle-type and neuronal-type nAChRs are functional in airway fibroblasts and HBE cells, that prior tobacco exposure does not appear to be an important variable in nAChR expression, and that distinct signaling pathways are observed in response to nicotine.

  15. Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis (Lepidoptera: Crambidae).

    Science.gov (United States)

    Xu, Gang; Wu, Shun-Fan; Teng, Zi-Wen; Yao, Hong-Wei; Fang, Qi; Huang, Jia; Ye, Gong-Yin

    2017-06-01

    Nicotinic acetylcholine receptors (nAChRs) are members of the cys-loop ligand-gated ion channel (cysLGIC) superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressalis, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs (cDNAs) via reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine α-type and three β-type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species. ©2016 The Authors Insect Science published by John Wiley & Sons Australia, Ltd on behalf of Institute of Zoology, Chinese Academy of Sciences.

  16. Acetylcholine Modulates the Hormones of the Growth Hormone/Insulinlike Growth Factor-1 Axis During Development in Mice.

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    Lecomte, Marie-José; Bertolus, Chloé; Ramanantsoa, Nélina; Saurini, Françoise; Callebert, Jacques; Sénamaud-Beaufort, Catherine; Ringot, Maud; Bourgeois, Thomas; Matrot, Boris; Collet, Corinne; Nardelli, Jeannette; Mallet, Jacques; Vodjdani, Guilan; Gallego, Jorge; Launay, Jean-Marie; Berrard, Sylvie

    2018-04-01

    Pituitary growth hormone (GH) and insulinlike growth factor (IGF)-1 are anabolic hormones whose physiological roles are particularly important during development. The activity of the GH/IGF-1 axis is controlled by complex neuroendocrine systems including two hypothalamic neuropeptides, GH-releasing hormone (GHRH) and somatostatin (SRIF), and a gastrointestinal hormone, ghrelin. The neurotransmitter acetylcholine (ACh) is involved in tuning GH secretion, and its GH-stimulatory action has mainly been shown in adults but is not clearly documented during development. ACh, together with these hormones and their receptors, is expressed before birth, and somatotroph cells are already responsive to GHRH, SRIF, and ghrelin. We thus hypothesized that ACh could contribute to the modulation of the main components of the somatotropic axis during development. In this study, we generated a choline acetyltransferase knockout mouse line and showed that heterozygous mice display a transient deficit in ACh from embryonic day 18.5 to postnatal day 10, and they recover normal ACh levels from the second postnatal week. This developmental ACh deficiency had no major impact on weight gain and cardiorespiratory status of newborn mice. Using this mouse model, we found that endogenous ACh levels determined the concentrations of circulating GH and IGF-1 at embryonic and postnatal stages. In particular, serum GH level was correlated with brain ACh content. ACh also modulated the levels of GHRH and SRIF in the hypothalamus and ghrelin in the stomach, and it affected the levels of these hormones in the circulation. This study identifies ACh as a potential regulator of the somatotropic axis during the developmental period.

  17. Brain regional acetylcholinesterase activity and muscarinic acetylcholine receptors in rats after repeated administration of cholinesterase inhibitors and its withdrawal

    International Nuclear Information System (INIS)

    Kobayashi, Haruo; Suzuki, Tadahiko; Sakamoto, Maki; Hashimoto, Wataru; Kashiwada, Keiko; Sato, Itaru; Akahori, Fumiaki; Satoh, Tetsuo

    2007-01-01

    Activity of acetylcholinesterase (AChE) and specific binding of [ 3 H]quinuclidinyl benzilate (QNB), [ 3 H]pirenzepine (PZP) and [ 3 H]AF-DX 384 to muscarinic acetylcholine receptor (mAChR) preparations in the striatum, hippocampus and cortex of rats were determined 1, 6 and 11 days after the last treatment with an organophosphate DDVP, a carbamate propoxur or a muscarinic agonist oxotremorine as a reference for 7 and 14 days. AChE activity was markedly decreased in the three regions 1 day after the treatment with DDVP for 7 and 14 days with a gradual recovery 6 to 11 days, and much less decreased 1, 6 and 11 days after the treatment with propoxur for 7 days but not for 14 days in the hippocampus and cortex. The binding of [ 3 H]-QNB, PZP and AF-DX 384 in the three regions was generally decreased by the treatment with DDVP for 7 and 14 days. Such down-regulations were generally restored 6 or 11 days after the treatment for 7 but not for 14 days. The down-regulation or up-regulation as measured by [ 3 H]-QNB, PZP and AF-DX 384 was observed 1, 6 or 11 days after treatment with propoxur for 7 days and/or 14 days. Repeated treatment with oxotremorine produced similar effects except AChE activity to DDVP. These results suggest that repeated inhibition of AChE activity may usually cause down-regulation of mAChRs with some exception in the hippocampus when a reversible antiChE propoxur is injected

  18. Acute desensitization of acetylcholine and endothelin-1 activated inward rectifier K+ current in myocytes from the cardiac atrioventricular node.

    Science.gov (United States)

    Choisy, Stéphanie C M; James, Andrew F; Hancox, Jules C

    2012-07-06

    The atrioventricular node (AVN) is a vital component of the pacemaker-conduction system of the heart, co-ordinating conduction of electrical excitation from cardiac atria to ventricles and acting as a secondary pacemaker. The electrical behaviour of the AVN is modulated by vagal activity via activation of muscarinic potassium current, IKACh. However, it is not yet known if this response exhibits 'fade' or desensitization in the AVN, as established for the heart's primary pacemaker--the sinoatrial node. In this study, acute activation of IKACh in rabbit single AVN cells was investigated using whole-cell patch clamp at 37 °C. 0.1-1 μM acetylcholine (ACh) rapidly activated a robust IKACh in AVN myocytes during a descending voltage-ramp protocol. This response was inhibited by tertiapin-Q (TQ; 300 nM) and by the M2 muscarinic ACh receptor antagonist AFDX-116 (1 μM). During sustained ACh exposure the elicited IKACh exhibited bi-exponential fade (τf of 2.0 s and τs 76.9 s at -120 mV; 1 μM ACh). 10 nM ET-1 elicited a current similar to IKACh, which faded with a mono-exponential time-course (τ of 52.6 s at -120 mV). When ET-1 was applied following ACh, the ET-1 activated response was greatly attenuated, demonstrating that ACh could desensitize the response to ET-1. For neither ACh nor ET-1 was the rate of current fade dependent upon the initial response magnitude, which is inconsistent with K+ flux mediated changes in electrochemical driving force as the underlying mechanism. Collectively, these findings demonstrate that TQ sensitive inwardly rectifying K+ current in cardiac AVN cells, elicited by M2 muscarinic receptor or ET-1 receptor activation, exhibits fade due to rapid desensitization. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Induced formation and maturation of acetylcholine receptor clusters in a defined 3D bio-artificial muscle.

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    Wang, Lin; Shansky, Janet; Vandenburgh, Herman

    2013-12-01

    Dysfunction of the neuromuscular junction is involved in a wide range of muscular diseases. The development of neuromuscular junction through which skeletal muscle is innervated requires the functional modulation of acetylcholine receptor (AchR) clustering on myofibers. However, studies on AchR clustering in vitro are mostly done on monolayer muscle cell culture, which lacks a three-dimensional (3D) structure, a prominent limitation of the two-dimensional (2D) system. To enable a better understanding on the structure-function correlation underlying skeletal muscle innervation, a muscle system with a well-defined geometry mimicking the in vivo muscular setting is needed. Here, we report a 3D bio-artificial muscle (BAM) bioengineered from green fluorescent protein-transduced C3H murine myoblasts as a novel in vitro tissue-based model for muscle innervation studies. Our cell biological and molecular analysis showed that this BAM is structurally similar to in vivo muscle tissue and can reach the perinatal differentiation stage, higher than does 2D culture. Effective clustering and morphological maturation of AchRs on BAMs induced by agrin and laminin indicate the functional activity and plasticity of this BAM system toward innervation. Taken together, our results show that the BAM provides a favorable 3D environment that at least partially recapitulates real physiological skeletal muscle with regard to innervation. With a convenience of fabrication and manipulation, this 3D in vitro system offers a novel model for studying mechanisms underlying skeletal muscle innervation and testing therapeutic strategies for relevant nervous and muscular diseases.

  20. The Dinoflagellate Toxin 20-Methyl Spirolide-G Potently Blocks Skeletal Muscle and Neuronal Nicotinic Acetylcholine Receptors

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    Aurélie Couesnon

    2016-08-01

    Full Text Available The cyclic imine toxin 20-methyl spirolide G (20-meSPX-G, produced by the toxigenic dinoflagellate Alexandrium ostenfeldii/Alexandrium peruvianum, has been previously reported to contaminate shellfish in various European coastal locations, as revealed by mouse toxicity bioassay. The aim of the present study was to determine its toxicological profile and its molecular target selectivity. 20-meSPX-G blocked nerve-evoked isometric contractions in isolated mouse neuromuscular preparations, while it had no action on contractions elicited by direct electrical stimulation, and reduced reversibly nerve-evoked compound muscle action potential amplitudes in anesthetized mice. Voltage-clamp recordings in Xenopus oocytes revealed that 20-meSPX-G potently inhibited currents evoked by ACh on Torpedo muscle-type and human α7 nicotinic acetylcholine receptors (nAChR, whereas lower potency was observed in human α4β2 nAChR. Competition-binding assays showed that 20-meSPX-G fully displaced [3H]epibatidine binding to HEK-293 cells expressing the human α3β2 (Ki = 0.040 nM, whereas a 90-fold lower affinity was detected in human α4β2 nAChR. The spirolide displaced [125I]α-bungarotoxin binding to Torpedo membranes (Ki = 0.028 nM and in HEK-293 cells expressing chick chimeric α7-5HT3 nAChR (Ki = 0.11 nM. In conclusion, this is the first study to demonstrate that 20-meSPX-G is a potent antagonist of nAChRs, and its subtype selectivity is discussed on the basis of molecular docking models.

  1. Gating at the mouth of the acetylcholine receptor channel: energetic consequences of mutations in the alphaM2-cap.

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    Pallavi A Bafna

    2008-06-01

    Full Text Available Gating of nicotinic acetylcholine receptors from a C(losed to an O(pen conformation is the initial event in the postsynaptic signaling cascade at the vertebrate nerve-muscle junction. Studies of receptor structure and function show that many residues in this large, five-subunit membrane protein contribute to the energy difference between C and O. Of special interest are amino acids located at the two transmitter binding sites and in the narrow region of the channel, where CO gating motions generate a lowhigh change in the affinity for agonists and in the ionic conductance, respectively. We have measured the energy changes and relative timing of gating movements for residues that lie between these two locations, in the C-terminus of the pore-lining M2 helix of the alpha subunit ('alphaM2-cap'. This region contains a binding site for non-competitive inhibitors and a charged ring that influences the conductance of the open pore. alphaM2-cap mutations have large effects on gating but much smaller effects on agonist binding, channel conductance, channel block and desensitization. Three alphaM2-cap residues (alphaI260, alphaP265 and alphaS268 appear to move at the outset of channel-opening, about at the same time as those at the transmitter binding site. The results suggest that the alphaM2-cap changes its secondary structure to link gating motions in the extracellular domain with those in the channel that regulate ionic conductance.

  2. Selective Activation of M4 Muscarinic Acetylcholine Receptors Reverses MK-801-Induced Behavioral Impairments and Enhances Associative Learning in Rodents

    Science.gov (United States)

    2015-01-01

    Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders. PMID:25137629

  3. Protective effect of an alpha 7 nicotinic acetylcholine receptor agonist against enterovirus 71 infection in neuronal cells.

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    Song, Feng Xia; Zhao, Lin Qing; Zhu, Ru Nan; Song, Qin Wei; Deng, Jie; Tian, Run; Wang, Fang; Qian, Yuan

    2018-01-01

    Enterovirus 71, as one of the dominant pathogens associated with severe hand, foot, and mouth disease, has been well reported to trigger severe neurological symptoms among young children over the last decade, particularly among children in the Asia-Pacific region. To date, no effective antiviral agent has been developed for the treatment of severe enterovirus 71 infection. PNU-282987, a selective alpha 7 nicotinic acetylcholine receptor (α7nAChR) agonist, has been reported to have a neuroprotective effect by participating in inflammatory regulation in previous studies. Therefore, in the present study, we aimed to assess the cell-protective effect of PNU-282987 against enterovirus 71 infection in neuronal cells, and to discuss potential mechanisms underlying this cell-protective effect in order to elucidate the potential impact of such agonists in the treatment of neurotropic viral infection. We observed that treatment with PNU-282987 improved cell viability and inhibited viral replication in enterovirus 71-infected SH-SY5Y cells. Further investigation revealed that inhibition of enterovirus 71 production by PNU-282987 is likely associated with events of RNA replication, and that increased levels of INF mRNA and its downstream antiviral proteins stimulated by the JAK-STAT2 pathway may contribute to the antiviral effect of PNU-282987. Moreover, our findings suggest that both the antiviral and anti-inflammatory effects of PNU-282987 may contribute to the neural protective effect of the drug in enterovirus 71-infected cells. Taken together, the results suggest that selective α7nAChR agonists may represent viable candidates for future therapeutic treatment of severe enterovirus 71 infection, and for other cases of neurotropic viral infection. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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    Igor E. Kasheverov

    2015-03-01

    Full Text Available 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 chicken α7 ligand-binding and glycine receptor transmembrane domains or on rat α4β2 nAChR expressed in Xenopus oocytes revealed no action of 6-BHP. However, in radioligand analysis, 6-BHP competed with radioiodinated α-bungarotoxin for binding to human α7 nAChR expressed in GH4C1 cells (IC50 23 ± 1 μM, but showed no competition on muscle-type nAChR from Torpedo californica. In Ca2+-imaging experiments on the human α7 nAChR expressed in the Neuro2a cells, 6-BHP in the presence of PNU120596 behaved as an agonist (EC50 ~80 μM. To the best of our knowledge, 6-BHP is the first low-molecular weight compound from marine source which is an agonist of the nAChR subtype. This may have physiological importance because H. crassicornis, with its simple and tractable nervous system, is a convenient model system for studying the learning and memory processes.

  5. The beneficial in vitro effects of lovastatin and chelerythrine on relaxatory response to acetylcholine in the perfused mesentric bed isolated from diabetic rats.

    Science.gov (United States)

    Fatehi-Hassanabad, Zahra; Imen-Shahidi, Mohsen; Fatehi, Mohammad; Farrokhfall, Khadijeh; Parsaeei, Heidar

    2006-03-27

    Diabetes mellitus is associated with an increased risk of cardiovascular disease. Endothelial dysfunction (i.e. decreased endothelium-dependent vasorelaxation) plays a key role in the pathogenesis of diabetic vascular disease. The present study was undertaken to determine whether diabetes induced by streptozotocin alters mesenteric responses to vasodilators and, if so, to study the acute in vitro effects of lovastatin and chelerythrine. Endothelial function was assessed in constantly perfused preparation removed from rats, 12 weeks after treatment with either saline or streptozotocin (45 mg/kg, intraperitoneally). In pre-contracted mesenteric beds (with 100 microM phenylephrine) removed from diabetic rats, the concentration response curve to acetylcholine, but not to sodium nitroprusside, was significantly reduced. Perfusion with lovastatin (10 microM for 20 min) or chelerythrine (1 microM for 20 min) significantly improved the acetylcholine-mediated relaxation in preparations removed from diabetic but not control rats. Pre-incubation of tissue with N(G)-nitro-L-argenine methyl ester hydrochloride (10 microM for 20 min) inhibited the beneficial effect of lovastatin but not chelerythrine. Pre-treatment of tissue with indomethacin (10 microM for 20 min) did not modify the effects of lovastatin or chelerythrine on acetylcholine responses. The present results demonstrate that endothelial dysfunction induced by diabetes (in a resistant vasculature, such as rat mesenteric bed) may be improved by an acute exposure to either lovastatin or chelerythrine. Furthermore, our results suggest that the beneficial effect of lovastatin is mediated via the nitric oxide pathway.

  6. The role of nicotinic acetylcholine and opioid systems of the ventral orbital cortex in modulation of formalin-induced orofacial pain in rats.

    Science.gov (United States)

    Yousofizadeh, Shahnaz; Tamaddonfard, Esmaeal; Farshid, Amir Abbas

    2015-07-05

    Nicotinic acetylcholine and opioid receptors are involved in modulation of pain. In the present study, we investigated the effects of microinjection of nicotinic acetylcholine and opioid compounds into the ventral orbital cortex (VOC) on the formalin-induced orofacial pain in rats. For this purpose, two guide cannulas were placed into the left and right sides of the VOC of the brain. Orofacial pain was induced by subcutaneous injection of a diluted formalin solution (50μl, 1.5%) into the right vibrissa pad and face rubbing durations were recorded at 3-min blocks for 45min. Formalin produced a marked biphasic pain response (first phase: 0-3min and second phase: 15-33min). Epibatidine (a nicotinic receptor agonist) at doses of 0.05, 0.1 and 0.2μg/site, morphine (an opioid receptor agonist) at doses of 0.5, 1 and 2μg/site and their sub-analgesic doses (0.025μg/site epibatidine with 0.25μg/site morphine) combination treatment suppressed the second phase of pain. The antinociceptive effect induced by 0.2μg/site of epibatidine, but not morphine (2μg/site), was prevented by 2μg/site of mecamylamine (a nicotinic receptor antagonist). Naloxone (an opioid receptor antagonist) at a dose of 2μg/site prevented the antinociceptive effects induced by 2μg/site of morphine and 0.2μg/site of epibatidine. No above-mentioned chemical compounds affected locomotor activity. These results showed that at the VOC level, epibatidine and morphine produced antinociception. In addition, opioid receptor might be involved in epibatidine-induced antinociception, but the antinociception induced by morphine was not mediated through nicotinic acetylcholine receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Extracellular dopamine, acetylcholine, and activation of dopamine D1 and D2 receptors after selective breeding for cocaine self-administration in rats.

    Science.gov (United States)

    Xu, Haiyang; Das, Sasmita; Sturgill, Marc; Hodgkinson, Colin; Yuan, Qiaoping; Goldman, David; Grasing, Kenneth

    2017-08-01

    The low self-administration (LS)/Kgras (LS) and high self-administration (HS)/Kgras (HS) rat lines were generated by selective breeding for low- and high-intravenous cocaine self-administration, respectively, from a common outbred Wistar stock (Crl:WI). This trait has remained stable after 13 generations of breeding. The objective of the present study is to compare cocaine preference, neurotransmitter release, and dopamine receptor activation in LS and HS rats. Levels of dopamine, acetylcholine, and cocaine were measured in the nucleus accumbens (NA) shell of HS and LS rats by tandem mass spectrometry of microdialysates. Cocaine-induced locomotor activity and conditioned-place preference were compared between LS and HS rats. HS rats displayed greater conditioned-place preference scores compared to LS and reduced basal extracellular concentrations of dopamine and acetylcholine. However, patterns of neurotransmitter release did not differ between strains. Low-dose cocaine increased locomotor activity in LS rats, but not in HS animals, while high-dose cocaine augmented activity only in HS rats. Either dose of cocaine increased immunoreactivity for c-Fos in the NA shell of both strains, with greater elevations observed in HS rats. Activation identified by cells expressing both c-Fos and dopamine receptors was generally greater in the HS strain, with a similar pattern for both D1 and D2 dopamine receptors. Diminished levels of dopamine and acetylcholine in the NA shell, with enhanced cocaine-induced expression of D1 and D2 receptors, are associated with greater rewarding effects of cocaine in HS rats and an altered dose-effect relationship for cocaine-induced locomotor activity.

  8. Retigabine diminishes the effects of acetylcholine, adrenaline and adrenergic agonists on the spontaneous activity of guinea pig smooth muscle strips in vitro.

    Science.gov (United States)

    Apostolova, Elisaveta; Zagorchev, Plamen; Kokova, Vesela; Peychev, Lyudmil

    2017-03-01

    The aim of this study is to evaluate the effect of retigabine on the smooth muscle response to acetylcholine, adrenaline, α-and β-adrenoceptor agonists. We studied the change in the spontaneous smooth muscle contraction of guinea pig gastric corpus strips before and after 20-min treatment with 2μM retigabine. We also evaluated the effect of retigabine on the smooth muscle response to 10μM acetylcholine, 1 and 10μM adrenaline, 1μM methoxamine, 0.1μM p-iodoclonidine and 10μM isoproterenol. We observed a significant reduction in the effects of all studied mediators and agonists when they were added to organ baths in the presence of retigabine. Retigabine diminished the effect of acetylcholine on the spontaneous smooth muscle activity. The effect was fully antagonized by XE-991 (Kv7 channel blocker), which supports our hypothesis about the role of KCNQ channels in the registered changes. The increase in the contraction force after adding of 1μM adrenaline, methoxamine, and 0.1μM p-iodoclonidine was also significantly smaller in presence of retigabine. However, comparing the effect of 10μM adrenaline on the contractility before and after treatment with retigabine, we observed increased contractility when retigabine was present in the organ baths. A possible explanation for the observed diminished effects of mediators and receptor agonists is that the effect of retigabine on smooth muscle contractility is complex. The membrane hyperpolarization, the interaction between Kv7 channels and adrenoceptors, and the influence on signaling pathways may contribute to the summary smooth muscle response. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

    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......AChR subtype/s involved remains unknown. In this study, we systematically compared the effects of non-selective and selective nicotinic agonists and antagonists in two different tests for antidepressant effects in mice: the tail suspension test and the forced swim test. Compounds: nicotine, RJR-2403 (alpha4...

  10. Regional distribution of muscarinic acetylcholine receptors in the telencephalon of the pigeon (Columba livia f. domestica). [Use of TH-quinuclidinylbenzylate

    Energy Technology Data Exchange (ETDEWEB)

    Waechtler, K.

    1985-01-01

    The distribution of muscarinic acetylcholine receptors was studied autoradiographically in croystat sections of the pigeon telencephalon using TH-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.

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

    DEFF Research Database (Denmark)

    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...... or indirectly involved in acute stress regulation after a single dose of ispronicline, supports earlier studies that the α4β2 receptors are strongly involved in nicotine-dependent activation of the hypothalamo-pituitary adrenocortical axis....

  12. Transcriptomic effects of depleted uranium on acetylcholine and cholesterol metabolisms in Alzheimer's disease model; Effets transcriptomiques de l'uranium appauvri sur les metabolismes de l'acetylcholine et du cholesterol chez un modele de maladie d'Alzheimer

    Energy Technology Data Exchange (ETDEWEB)

    Lestaevel, Ph.; Bensoussan, H.; Racine, R.; Airault, F.; Gourmelon, P.; Souidi, M. [Direction de la radioprotection de l' Homme, service de radiobiologie et d' epidemiologie, laboratoire de radiotoxicologie experimentale, institut de radioprotection et de surete nucleaire, BP no 17, 92262 Fontenay-aux-Roses cedex (France)

    2011-02-15

    Some heavy metals, or aluminium, could participate in the development of Alzheimer disease (AD). Depleted uranium (DU), another heavy metal, modulates the cholinergic system and the cholesterol metabolism in the brain of rats, but without neurological disorders. The aim of this study was to determine what happens in organisms exposed to DU that will/are developing the AD. This study was thus performed on a transgenic mouse model for human amyloid precursor protein (APP), the Tg2576 strain. The possible effects of DU through drinking water (20 mg/L) over an 8-month period were analyzed on acetylcholine and cholesterol metabolisms at gene level in the cerebral cortex. The mRNA levels of choline acetyl transferase (ChAT) vesicular acetylcholine transporter (VAChT) and ATP-binding cassette transporter A1 (ABC A1) decreased in control Tg2576 mice in comparison with wild-type mice (respectively -89%, -86% and -44%, p < 0.05). Chronic exposure of Tg2576 mice to DU increased mRNA levels of ChAT (+189%, p < 0.05), VAChT (+120%, p < 0.05) and ABC A1 (+52%, p < 0.05) compared to control Tg2576 mice. Overall, these modifications of acetylcholine and cholesterol metabolisms did not lead to increased disturbances that are specific of AD, suggesting that chronic DU exposure did not worsen the pathology in this experimental model. (authors)

  13. Human α4β2 nicotinic acetylcholine receptor as a novel target of oligomeric α-synuclein.

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    Full Text Available Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5'-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM analyses, we find that only large (>4 nm oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD.

  14. The Distribution of Charged Amino Acid Residues and the Ca2+ Permeability of Nicotinic Acetylcholine Receptors: A Predictive Model

    Directory of Open Access Journals (Sweden)

    Sergio Fucile

    2017-05-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are cation-selective ligand-gated ion channels exhibiting variable Ca2+ permeability depending on their subunit composition. The Ca2+ permeability is a crucial functional parameter to understand the physiological role of nAChRs, in particular considering their ability to modulate Ca2+-dependent processes such as neurotransmitter release. The rings of extracellular and intracellular charged amino acid residues adjacent to the pore-lining TM2 transmembrane segment have been shown to play a key role in the cation selectivity of these receptor channels, but to date a quantitative relationship between these structural determinants and the Ca2+ permeability of nAChRs is lacking. In the last years the Ca2+ permeability of several nAChR subtypes has been experimentally evaluated, in terms of fractional Ca2+ current (Pf, i.e., the percentage of the total current carried by Ca2+ ions. In the present study, the available Pf-values of nAChRs are used to build a simplified modular model describing the contribution of the charged residues in defined regions flanking TM2 to the selectivity filter controlling Ca2+ influx. This model allows to predict the currently unknown Pf-values of existing nAChRs, as well as the hypothetical Ca2+ permeability of subunit combinations not able to assemble into functional receptors. In particular, basing on the amino acid sequences, a Pf > 50% would be associated with homomeric nAChRs composed by different α subunits, excluding α7, α9, and α10. Furthermore, according to the model, human α7β2 receptors should have Pf-values ranging from 3.6% (4:1 ratio to 0.1% (1:4 ratio, much lower than the 11.4% of homomeric α7 nAChR. These results help to understand the evolution and the function of the large diversity of the nicotinic receptor family.

  15. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  16. Synthesis and evaluation of new imaging agent for central nicotinic acetylcholine receptor {alpha}{sub 7} subtype

    Energy Technology Data Exchange (ETDEWEB)

    Ogawa, Mikako [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu (Japan); Nishiyama, Shingo; Tsukada, Hideo [PET Center, Central Research Laboratory, Hamamatsu Photonics K.K., Hamamatsu (Japan); Hatano, Kentaro [National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu (Japan); Fuchigami, Takeshi [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu (Japan); Yamaguchi, Hiroshi [National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu (Japan); Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu (Japan); Matsushima, Yoshitaka [Department of Chemistry, Hamamatsu University School of Medicine, Hamamatsu (Japan); Ito, Kengo [National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu (Japan); Magata, Yasuhiro [Photon Medical Research Center, Hamamatsu University School of Medicine, Hamamatsu (Japan); Molecular Imaging Frontier Research Center, Hamamatsu University School of Medicine, Hamamatsu (Japan)], E-mail: magata@hama-med.ac.jp

    2010-04-15

    Introduction: The nicotinic acetylcholine receptor (nAChR) {alpha}7 subtype ({alpha}{sub 7} nAChR) is one of the major nAChR subtypes in the brain. We synthesized C-11 labeled {alpha}{sub 7} nAChR ligands, (R)-2-[{sup 11}C]methylamino-benzoic acid 1-aza-bicyclo[2.2.2]oct-3-yl ester ([{sup 11}C](R)-MeQAA) and its isomer (S)-[{sup 11}C]MeQAA, for in vivo investigation with positron emission tomography (PET). Then, the potential of (R)- and (S)-[{sup 11}C]MeQAA for in vivo imaging of {alpha}{sub 7} nAChR in the brain was evaluated in mice and monkeys. Methods: The binding affinity for {alpha}{sub 7} 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 {alpha}{sub 7} nAChR was 41 and 182 nM for (R)- and (S)-MeQAA, respectively. The initial uptake in the mouse brain was high ([{sup 11}C](R)-MeQAA: 7.68 and [{sup 11}C](S)-MeQAA: 6.65 %dose/g at 5 min). The clearance of [{sup 11}C](R)-MeQAA was slow in the hippocampus ({alpha}{sub 7} nAChR-rich region) but was rapid in the cerebellum ({alpha}{sub 7} nAChR-poor region). On the other hand, the clearance was fast for [{sup 11}C](S)-MeQAA in all regions. The brain uptake of [{sup 11}C](R)-MeQAA was decreased by methyllycaconitine ({alpha}{sub 7} nAChR antagonist) treatment. In monkeys, {alpha}{sub 7} 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 [{sup 11}C](R)-MeQAA, while the uptake was rather homogeneous for [{sup 11}C](S)-MeQAA. Conclusions: [{sup 11}C](R)-MeQAA was successfully synthesized and showed high uptake to the brain. However, since the in vivo selectivity for {alpha}{sub 7} nAChR was not enough, further PET kinetic analysis or structure optimization is needed for specific visualization of brain {alpha}{sub 7} nAChRs in vivo.

  17. Assessment of {alpha}7 nicotinic acetylcholine receptor availability in juvenile pig brain with [{sup 18}F]NS10743

    Energy Technology Data Exchange (ETDEWEB)

    Deuther-Conrad, Winnie; Fischer, Steffen; Hiller, Achim; Funke, Uta; Brust, Peter [Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmacy, Leipzig (Germany); Becker, Georg; Sabri, Osama [Univ. of Leipzig, Dept. of Nuclear Medicine, Leipzig (Germany); Cumming, Paul; Xiong, Guoming [Univ. of Munich, Dept. of Nuclear Medicine, Munich (Germany); Peters, Dan [NeuroSearch A/S, Ballerup (Denmark)

    2011-08-15

    To conduct a quantitative PET assessment of the specific binding sites in the brain of juvenile pigs for [{sup 18}F]NS10743, a novel diazabicyclononane derivative targeting {alpha}7 nicotinic acetylcholine receptors ({alpha}7 nAChRs). Dynamic PET recordings were made in isoflurane-anaesthetized juvenile pigs during 120 min after administration of [{sup 18}F]NS10743 under baseline conditions (n = 3) and after blocking of the {alpha}7 nAChR with NS6740 (3 mg.kg{sup -1} bolus + 1 mg.kg{sup -1}.h{sup -1} continuous infusion; n = 3). Arterial plasma samples were collected for determining the input function of the unmetabolized tracer. Kinetic analysis of regional brain time-radioactivity curves was performed, and parametric maps were calculated relative to arterial input. Plasma [{sup 18}F]NS10743 passed readily into the brain, with peak uptake occurring in {alpha}7 nAChR-expressing brain regions such as the colliculi, thalamus, temporal lobe and hippocampus. The highest SUV{sub max} was approximately 2.3, whereas the lowest uptake was in the olfactory bulb (SUV{sub max} 1.53 {+-} 0.32). Administration of NS6740 significantly decreased [{sup 18}F]NS10743 binding late in the emission recording throughout the brain, except in the olfactory bulb, which was therefore chosen as reference region for calculation of BP{sub ND}. The baseline BP{sub ND} ranged from 0.39 {+-} 0.08 in the cerebellum to 0.76 {+-} 0.07 in the temporal lobe. Pretreatment and constant infusion with NS6740 significantly reduced the BP{sub ND} in regions with high [{sup 18}F]NS10743 binding (temporal lobe -29%, p = 0.01; midbrain: -35%, p = 0.02), without significantly altering the BP{sub ND} in low binding regions (cerebellum: -16%, p = 0.2). This study confirms the potential of [{sup 18}F]NS10743 as a target-specific radiotracer for the molecular imaging of central {alpha}7 nAChRs by PET. (orig.)

  18. Synthesis and evaluation of [125I]I-TSA as a brain nicotinic acetylcholine receptor α7 subtype imaging agent

    International Nuclear Information System (INIS)

    Ogawa, Mikako; Tatsumi, Ryo; Fujio, Masakazu; Katayama, Jiro; Magata, Yasuhiro

    2006-01-01

    Introduction: Some in vitro investigations have suggested that the nicotinic acetylcholine receptor (nAChR) α 7 subtype is implicated in Alzheimer's disease, schizophrenia and others. Recently, we developed (R)-3'-(5-bromothiophen-2-yl)spiro[1-azabicyclo[2.2.2]octane-3,5'-[1',3'] oxazolidin]-2'-one (Br-TSA), which has a high affinity and selectivity for α 7 nAChRs. Therefore we synthesized (R)-3'-(5-[ 125 I]iodothiophen-2-yl)spiro[1-azabicyclo[2.2.2]octane-3,5'- [1',3']oxazolidin]-2'-one ([ 125 I]I-TSA) and evaluated its potential for the in vivo detection of α 7 nAChR in brain. Methods: In vitro binding affinity of I-TSA was measured in rat brain homogenates. Radioiodination was accomplished by a Br-I exchange reaction. Biodistribution studies were undertaken in mice by tail vein injection of [ 125 I]I-TSA. In vivo receptor blocking studies were carried out by treating mice with methyllycaconitine (MLA; 5 nmol/5 μl, i.c.v.) or nonradioactive I-TSA (50 μmol/kg, i.v.). Results: I-TSA exhibited a high affinity and selectivity for the α 7 nAChR (K i for α 7 nAChR=0.54 nM). Initial uptake in the brain was high (4.42 %dose/g at 5 min), and the clearance of radioactivity was relatively slow in the hippocampus (α 7 nAChR-rich region) and was rather rapid in the cerebellum (α 7 nAChR poor region). The hippocampus to cerebellum uptake ratio was 0.9 at 5 min postinjection, but it was increased to 1.8 at 60 min postinjection. Although the effect was not statistically significant, administration of I-TSA and MLA decreased the accumulation of radioactivity in hippocampus. Conclusion: Despite its high affinity and selectivity, [ 125 I]I-TSA does not appear to be a suitable tracer for in vivo α 7 nAChR receptor imaging studies due to its high nonspecific binding. Further structural optimization is needed

  19. Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

    Directory of Open Access Journals (Sweden)

    Field Linda M

    2011-05-01

    Full Text Available Abstract Background Myzus persicae is a globally important aphid pest with a history of developing resistance to insecticides. Unusually, neonicotinoids have remained highly effective as control agents despite nearly two decades of steadily increasing use. In this study, a clone of M. persicae collected from southern France was found, for the first time, to exhibit sufficiently strong resistance to result in loss of the field effectiveness of neonicotinoids. Results Bioassays, metabolism and gene expression studies implied the presence of two resistance mechanisms in the resistant clone, one based on enhanced detoxification by cytochrome P450 monooxygenases, and another unaffected by a synergist that inhibits detoxifying enzymes. Binding of radiolabeled imidacloprid (a neonicotinoid to whole body membrane preparations showed that the high affinity [3H]-imidacloprid binding site present in susceptible M. persicae is lost in the resistant clone and the remaining lower affinity site is altered compared to susceptible clones. This confers a significant overall reduction in binding affinity to the neonicotinoid target: the nicotinic acetylcholine receptor (nAChR. Comparison of the nucleotide sequence of six nAChR subunit (Mpα1-5 and Mpβ1 genes from resistant and susceptible aphid clones revealed a single point mutation in the loop D region of the nAChR β1 subunit of the resistant clone, causing an arginine to threonine substitution (R81T. Conclusion Previous studies have shown that the amino acid at this position within loop D is a key determinant of neonicotinoid binding to nAChRs and this amino acid change confers a vertebrate-like character to the insect nAChR receptor and results in reduced sensitivity to neonicotinoids. The discovery of the mutation at this position and its association with the reduced affinity of the nAChR for imidacloprid is the first example of field-evolved target-site resistance to neonicotinoid insecticides and also

  20. Studies for transitional changes of the muscarinic acetylcholine receptor and mRNA distribution by focal ischemia using nuclear medicine

    Energy Technology Data Exchange (ETDEWEB)

    Kuji, Ichiei [Kanazawa Univ. (Japan). School of Medicine

    1994-04-01

    Assessing stress-induced brain receptor responses is important in understanding clinical brain receptor images for nuclear medicine. It is known that cholinergic neurons are decreased by Alzheimer`s disease and that there is a close relationship between cholinergic neurons and muscarinic acetylcholine receptors (mAchR). Thus, this study assessed the response of mAchR to focal ischemia using infarction model rats (prepared by middle cerebral artery occlusion) and sham-operated rats. In the same rats, three kinds of images -- ex vivo regional cerebral blood flow (rCBF) images with {sup 99m}Tc-hexametyl-propyleneamine oxime ({sup 99m}Tc-HMPAO), in vitro mAchR binding images with [{sup 3}H] quinuclidinyl benzilate ({sup 3}H-QNB), and mAchR-mRNA images by in situ hybridization method using {sup 35}S-labeled-oligonucleotide probes specific for mAchR gene subtypes of m1 to m5 -- were obtained in acute and chronic phases. Each image datum was digitalized and assessed semi-quantitatively. There were significant changes in global distribution among rCBF, mAchR and mAchR-mRNAs. In the acute phase, there was no significant change in mAchR in the infarcted area, although rCBF markedly decreased. In the chronic phase, there was a significant decrease in mAchR in the infarct-sided thalamus, although there was no change in rCBF; and there was a significant decrease in mAchR of the infarct-sided substantia nigra in spite of increase in rCBF. In the acute phase, mAchR-mRNAs of the infarct-sided caudate-putamen was decreased, suggesting that the ability of cholinergic neuron to synthesize receptor protein had decreased in the acute phase. Because mAchR was not decreased in the acute phase, some viable neurons with no normal function may be preserved in the acute phase. These results were encouraging in understanding mAchR brain images of patients with memory disturbances such as cerebrovascular dementia and Alzheimer`s disease. (N.K.).

  1. SPET imaging of central muscarinic acetylcholine receptors with iodine-123 labelled E-IQNP and Z-IQNP

    Energy Technology Data Exchange (ETDEWEB)

    Nobuhara, K.; Farde, L.; Halldin, C.; Karlsson, P.; Swahn, C.G.; Olsson, H.; Sedvall, G. [Dept. of Clinical Neuroscience, Psychiatry Section, Karolinska Hospital, Stockholm (Sweden); Bergstroem, K.A. [Dept. of Clinical Physiology, Kuopio University Hospital, Kuopio (Finland); Larsson, S.A.; Schnell, P.-O. [Dept. of Radiation Physics, Karolinska Hospital, Stockholm (Sweden); McPherson, D.W. [Oak Ridge National Laboratory (ORNL), Nuclear Medicine Group, TN (United States); Savonen, A.; Hiltunen, J. [MAP Medical Technologies Oy, Tikkakoski (Finland)

    2001-01-01

    1-Azabicyclo[2.2.2]oct-3-yl {alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP) is a muscarinic acetylcholine receptor (mAChR) antagonist and the racemic ligand contains eight stereoisomers. In a single-photon emission tomography (SPET) study in monkeys we recently confirmed that [{sup 123}I]E-(R,R)-IQNP ([{sup 123}I]E-IQNP) is a radioligand with modest selectivity for the M{sub 1} and M{sub 4} subtypes, whereas [{sup 123}I]Z-(R,R)-IQNP ([{sup 123}I]Z-IQNP) is non-subtype selective. In the present SPET study, E- and Z-IQNP were examined in human subjects. SPET examination was performed on three male subjects after i.v. injection of [{sup 123}I]E-IQNP and in another three after i.v. injection of [{sup 123}I]Z-IQNP. The binding potential (BP) for [{sup 123}I]E-IQNP was calculated using several quantitative approaches with the cerebellum as a reference region. High-performance liquid chromatography was used to measure radioligand metabolism in plasma. Following [{sup 123}I]E-IQNP, the radioactivity was high in the neocortex and striatum, intermediate in the thalamus and low in the pons and cerebellum, which is consistent with the rank order for the regional density of M{sub 1} and M{sub 4} subtypes in vitro. For all regions, peak equilibrium was identified within the 48-h data acquisition. The simplified reference tissue approach using SPET data from 0 to 48 h was the most reliable in this limited series of subjects. Following injection of [{sup 123}I]Z-IQNP, radioactivity was high in the neocortex and striatum, intermediate in the thalamus and pons and low in the cerebellum, which is in agreement with the density of M{sub 1}, M{sub 2} and M{sub 4} subtypes as measured in vitro. Quantitative analyses provided indirect support for specific M{sub 2} binding of Z-IQNP in the cerebellum. The high selectivity of [{sup 123}I]E-IQNP for M{sub 1} and M{sub 4} receptors allowed the use of cerebellum as a reference region devoid of specific binding, and

  2. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  3. Effect of α{sub 7} nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    Energy Technology Data Exchange (ETDEWEB)

    Welch, Kevin D., E-mail: kevin.welch@ars.usda.gov [USDA/ARS Poisonous Plant Research Laboratory, 1150 E. 1400N., Logan, UT 84341 (United States); Pfister, James A. [USDA/ARS Poisonous Plant Research Laboratory, 1150 E. 1400N., Logan, UT 84341 (United States); Lima, Flavia G. [Federal University of Goías, School of Veterinary Medicine, Goiânia, Goías (Brazil); Green, Benedict T.; Gardner, Dale R. [USDA/ARS Poisonous Plant Research Laboratory, 1150 E. 1400N., Logan, UT 84341 (United States)

    2013-02-01

    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.

  4. The Sleep–Wake Cycle in the Nicotinic Alpha-9 Acetylcholine Receptor Subunit Knock-Out Mice

    Directory of Open Access Journals (Sweden)

    Natalia Madrid-López

    2017-10-01

    Full Text Available There is a neural matrix controlling the sleep–wake cycle (SWC embedded within high ranking integrative mechanisms in the central nervous system. Nicotinic alpha-9 acetylcholine receptor subunit (alpha-9 nAChR participate in physiological processes occurring in sensory, endocrine and immune systems. There is a relationship between the SWC architecture, body homeostasis and sensory afferents so that disruption of afferent signaling is expected to affect the temporal organization of sleep and wake states. The analysis of the SWC of 9 nAChR knock-out animals may help to reveal the contribution of alpha-9 nAChR to sleep chronobiological determinants. Here we explore the polysomnogram in chronically implanted alpha-9 nAChR knock-out (KO and wild-type (WT individuals of the hybrid CBA/Sv129 mouse strain. Records were obtained in isolation chambers under a stable 12:12 light:dark cycle (LD. To unmask the 24-h modulation of the SWC a skeleton photoperiod (SP protocol was performed. Under LD the daily quota (in % of wakefulness (W, NREM sleep and REM sleep obtained in KO and WT animals were 45, 48 and 7, and 46, 46 and 8 respectively. Both groups exhibit nocturnal phase preference of W as well as diurnal and unimodal phase preference of NREM and REM sleep. The acrophase mean angles of KO vs. WT genotypes were not different (Zeitgeber Time: 6.5 vs. 14.9 for W, 4.3 vs. 2.8 for NREM sleep and 5.3 vs. 3.4 for REM sleep, respectively. Transference to SP do not affect daily state quotas, phase preferences and acrophases among genotypes. Unmasking phenomena of the SWC such as wake increment during the rest phase under SP was evident only among WT mice suggesting the involvement of retinal structures containing alpha-9 nAChR in masking processes. Furthermore, KO animals exhibit longer NREM and REM sleep episodes that is independent of illumination conditions. Consolidated diurnal NREM sleep contributed to obtain higher values of NREM sleep delta-EEG activity

  5. Tropical Malaysians and temperate Koreans exhibit significant differences in sweating sensitivity in response to iontophoretically administered acetylcholine

    Science.gov (United States)

    Lee, Jeong-Beom; Bae, Jun-Sang; Matsumoto, Takaaki; Yang, Hun-Mo; Min, Young-Ki

    2009-03-01

    Natives of the tropics are able to tolerate high ambient temperatures. This results from their long-term residence in hot and often humid tropical climates. This study was designed to compare the peripheral mechanisms of thermal sweating in tropical natives with that of their temperate counterparts. Fifty-five healthy male subjects including 20 native Koreans who live in the temperate Korean climate (Temperate-N) and 35 native tropical Malaysian men that have lived all of their lives in Malaysia (Tropical-N) were enrolled in this study after providing written informed consent to participate. Quantitative sudomotor axon reflex testing after iontophoresis (2 mA for 5 min) with 10% acetylcholine (ACh) was used to determine directly activated (DIR) and axon reflex-mediated (AXR) sweating during ACh iontophoresis. The sweat rate, activated sweat gland density, sweat gland output per single gland activated, and oral and skin temperature changes were measured. The sweat onset time of AXR (nicotinic-receptor-mediated) was 56 s shorter in the Temperate-N than in the Tropical-N subjects ( P < 0.0001). The nicotinic-receptor-mediated sweating activity AXR (1), and the muscarinic-receptor-mediated sweating activity DIR, in terms of sweat volume, were 103% and 59% higher in the Temperate-N compared to the Tropical-N subjects ( P < 0.0001). The Temperate-N group also had a 17.8% ( P < 0.0001) higher active sweat gland density, 35.4% higher sweat output per gland, 0.24°C higher resting oral temperature, and 0.62°C higher resting forearm skin temperature compared to the Tropical-N subjects ( P < 0.01). ACh iontophoresis did not influence oral temperature, but increased skin temperature near where the ACh was administered, in both groups. These results suggest that suppressed thermal sweating in the Tropical-N subjects was, at least in part, due to suppressed sweat gland sensitivity to ACh through both recruitment of active sweat glands and the sweat gland output per each gland

  6. Ocular myasthenia gravis induced by human acetylcholine receptor ϵ subunit immunization in HLA DR3 transgenic mice.

    Science.gov (United States)

    Wu, Xiaorong; Tuzun, Erdem; Saini, Shamsher S; Wang, Jun; Li, Jing; Aguilera-Aguirre, Leopoldo; Huda, Ruksana; Christadoss, Premkumar

    2015-12-01

    Extraocular muscles (EOM) are preferentially involved in myasthenia gravis (MG) and acetylcholine receptor (AChR) antibody positive MG patients may occasionally present with isolated ocular symptoms. Although experimental autoimmune myasthenia gravis (EAMG) induced by whole AChR immunization closely mimics clinical and immunopathological aspects of MG, EOM are usually not affected. We have previously developed an EAMG model, which imitates EOM symptoms of MG by immunization of human leukocyte antigen (HLA) transgenic mice with α or γ-subunits of human AChR (H-AChR). To investigate the significance of the ϵ-subunit in ocular MG, we immunized HLA-DR3 and HLA-DQ8 transgenic mice with recombinant H-AChR ϵ-subunit expressed in Escherichia coli. HLA-DR3 transgenic mice showed significantly higher clinical ocular and generalized MG severity scores and lower grip strength values than HLA-DQ8 mice. H-AChR ϵ-subunit-immunized HLA-DR3 transgenic mice had higher serum anti-AChR antibody (IgG, IgG1, IgG2b, IgG2c and IgM) levels, neuromuscular junction IgG and complement deposit percentages than ϵ-subunit-immunized HLA-DQ8 transgenic mice. Control mice immunized with E. coli extract or complete Freund adjuvant (CFA) did not show clinical and immunopathological features of ocular and generalized EAMG. Lymph node cells of ϵ-subunit-immunized HLA-DR3 mice showed significantly higher proliferative responses than those of ϵ-subunit-immunized HLA-DQ8 mice, crude E. coli extract-immunized and CFA-immunized transgenic mice. Our results indicate that the human AChR ϵ-subunit is capable of inducing myasthenic muscle weakness. Diversity of the autoimmune responses displayed by mice expressing different HLA class II molecules suggests that the interplay between HLA class II alleles and AChR subunits might have a profound impact on the clinical course of MG. Copyright © 2015 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  7. Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

    Science.gov (United States)

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

    2016-01-01

    The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes.

  8. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    International Nuclear Information System (INIS)

    Bencherif, M.; Lukas, R.J.

    1991-01-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate [ 3 H-QNB]. The rank order potency of selective antagonists that inhibit specific 3 HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2[2-[(diethylamino)methyl]-1-[piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of 3 HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA

  9. Promoted neuronal differentiation after activation of alpha4/beta2 nicotinic acetylcholine receptors in undifferentiated neural progenitors.

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    Takeshi Takarada

    Full Text Available BACKGROUND: Neural progenitor is a generic term used for undifferentiated cell populations of neural stem, neuronal progenitor and glial progenitor cells with abilities for proliferation and differentiation. We have shown functional expression of ionotropic N-methyl-D-aspartate (NMDA and gamma-aminobutyrate type-A receptors endowed to positively and negatively regulate subsequent neuronal differentiation in undifferentiated neural progenitors, respectively. In this study, we attempted to evaluate the possible functional expression of nicotinic acetylcholine receptor (nAChR by undifferentiated neural progenitors prepared from neocortex of embryonic rodent brains. METHODOLOGY/PRINCIPAL FINDINGS: Reverse transcription polymerase chain reaction analysis revealed mRNA expression of particular nAChR subunits in undifferentiated rat and mouse progenitors prepared before and after the culture with epidermal growth factor under floating conditions. Sustained exposure to nicotine significantly inhibited the formation of neurospheres composed of clustered proliferating cells and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction activity at a concentration range of 1 µM to 1 mM without affecting cell survival. In these rodent progenitors previously exposed to nicotine, marked promotion was invariably seen for subsequent differentiation into cells immunoreactive for a neuronal marker protein following the culture of dispersed cells under adherent conditions. Both effects of nicotine were significantly prevented by the heteromeric α4β2 nAChR subtype antagonists dihydro-β-erythroidine and 4-(5-ethoxy-3-pyridinyl-N-methyl-(3E-3-buten-1-amine, but not by the homomeric α7 nAChR subtype antagonist methyllycaconitine, in murine progenitors. Sustained exposure to nicotine preferentially increased the expression of Math1 among different basic helix-loop-helix proneural genes examined. In undifferentiated progenitors from embryonic mice

  10. Ligand binding and functional characterization of muscarinic acetylcholine receptors on the TE671/RD human cell line

    Energy Technology Data Exchange (ETDEWEB)

    Bencherif, M.; Lukas, R.J. (Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (USA))

    1991-06-01

    Cells of the TE671/RD human clonal line express a finite number ((Bmax) of about 350 fmol/mg of membrane protein) of apparently noninteracting, high-affinity binding sites (KD of 0.07 nM and a Hill coefficient close to unity, nH = 0.94) for the muscarinic acetylcholine receptor (mAChR) radio antagonist, tritium-labeled quinuclidinyl benzilate ({sup 3}H-QNB). The rank order potency of selective antagonists that inhibit specific {sup 3}HQNB binding is: atropine greater than 4-DAMP (4-diphenylacetoxy-N-methylpiperidine methiodide) greater than pirenzepine greater than methoctramine greater than AFDx-116 (11-2(2-((diethylamino)methyl)-1-(piperidinyl) acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepin-6-one). Functional studies indicate that phosphoinositide (PIns) hydrolysis in TE671/RD cells is increased by carbachol (EC50 of 10 microM), but not by nicotine (to concentrations as high as 1 mM). Agonist-stimulated PIns metabolism is inhibited by antagonists with the same rank order potency as for inhibition of {sup 3}HQNB binding. Functional responses are augmented in the presence of a nonhydrolyzable GTP analog, are strongly inhibited after 24-hr exposure to cholera toxin, but are only slightly inhibited after long-term exposure to pertussis toxin or forskolin. These studies identify a pharmacologically-defined M3-subtype of mAChR strongly coupled via a cholera toxin-sensitive mechanism to PIns hydrolysis in these cells. Within 1 hr of treatment of TE671/RD cells with 1 mM dibutyryl cyclic AMP or with 10 microM phorbol-12-myristate-13-acetate (PMA), there is a 30 to 50% decrease in carbachol-stimulated PIns responsiveness that recovers to control values after 5 days of continued drug treatment. However, a comparable and more persistent inhibition of mAChR function is observed on cell treatment with 20 nM PMA.

  11. The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography

    International Nuclear Information System (INIS)

    Weinberger, D.R.; Gibson, R.; Coppola, R.; Jones, D.W.; Molchan, S.; Sunderland, T.; Berman, K.F.; Reba, R.C.

    1991-01-01

    A high-affinity muscarinic receptor antagonist, 123IQNB (3-quinuclidinyl-4-iodobenzilate labeled with iodine 123), was used with single photon emission computed tomography to image muscarinic acetylcholine receptors in 14 patients with dementia and in 11 healthy controls. High-resolution single photon emission computed tomographic scanning was performed 21 hours after the intravenous administration of approximately 5 mCi of IQNB. In normal subjects, the images of retained ligand showed a consistent regional pattern that correlated with postmortem studies of the relative distribution of muscarinic receptors in the normal human brain, having high radioactivity counts in the basal ganglia, occipital cortex, and insular cortex, low counts in the thalamus, and virtually no counts in the cerebellum. Eight of 12 patients with a clinical diagnosis of Alzheimer's disease had obvious focal cortical defects in either frontal or posterior temporal cortex. Both patients with a clinical diagnosis of Pick's disease had obvious frontal and anterior temporal defects. A region of interest statistical analysis of relative regional activity revealed a significant reduction bilaterally in the posterior temporal cortex of the patients with Alzheimer's disease compared with controls. This study demonstrates the practicability of acetylcholine receptor imaging with 123IQNB and single photon emission computed tomography. The data suggest that focal abnormalities in muscarinic binding in vivo may characterize some patients with Alzheimer's disease and Pick's disease, but further studies are needed to address questions about partial volume artifacts and receptor quantification

  12. Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

    Science.gov (United States)

    Sundberg, Sofie Charlotte; Lindström, Sarah Helen; Sanchez, Gonzalo Manuel; Granseth, Björn

    2018-01-01

    The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal. © 2017 Wiley Periodicals, Inc.

  13. The association between the nicotinic acetylcholine receptor α4 subunit gene (CHRNA4 rs1044396 and Internet gaming disorder in Korean male adults.

    Directory of Open Access Journals (Sweden)

    Jo-Eun Jeong

    Full Text Available The primary aim of this study was to investigate the genetic predisposition of Internet gaming disorder (IGD, and the secondary aim was to compare the results to those of alcohol dependence (AD. Two independent case-control studies were conducted. A total of 30 male participants with IGD, diagnosed according to the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5 criteria, and 30 sex-matched controls participated in study 1. We designed targeted exome sequencing (TES to test for 72 candidate genes that have been implicated in the pathogenesis of addiction. The genes included seven neurotransmitter (dopamine, serotonin, glutamate, r-aminobutyric acid (GABA, norepinephrine, acetylcholine, and opioid system genes. A total of 31 male in-patients with AD and 29 normal male controls (NC were enrolled in study 2. The same 72 genes included in study 1 and ten additional genes related to alcohol-metabolic enzyme were selected as the target genes, and we identified the genetic variants using the same method (TES. The IGD group had a lower frequency of the T allele of rs1044396 in the nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4, and this variant represents a protective allele against IGD. However, we did not find a significant difference in the polymorphisms of the 72 genes that encode neurotransmitter systems between the AD and NC groups. This study demonstrated that rs1044396 of CHRNA4 was significantly associated with IGD.

  14. The dual orexin receptor antagonist, DORA-22, lowers histamine levels in the lateral hypothalamus and prefrontal cortex without lowering hippocampal acetylcholine.

    Science.gov (United States)

    Yao, Lihang; Ramirez, Andres D; Roecker, Anthony J; Fox, Steven V; Uslaner, Jason M; Smith, Sean M; Hodgson, Robert; Coleman, Paul J; Renger, John J; Winrow, Christopher J; Gotter, Anthony L

    2017-07-01

    Chronic insomnia is defined as a persistent difficulty with sleep initiation maintenance or non-restorative sleep. The therapeutic standard of care for this condition is treatment with gamma-aminobutyric acid (GABA) A receptor modulators, which promote sleep but are associated with a panoply of side effects, including cognitive and memory impairment. Dual orexin receptor antagonists (DORAs) have recently emerged as an alternative therapeutic approach that acts via a distinct and more selective wake-attenuating mechanism with the potential to be associated with milder side effects. Given their distinct mechanism of action, the current work tested the hypothesis that DORAs and GABA A receptor modulators differentially regulate neurochemical pathways associated with differences in sleep architecture and cognitive performance induced by these pharmacological mechanisms. Our findings showed that DORA-22 suppresses the release of the wake neurotransmitter histamine in the lateral hypothalamus, prefrontal cortex, and hippocampus with no significant alterations in acetylcholine levels. In contrast, eszopiclone, commonly used as a GABA A modulator, inhibited acetylcholine secretion across brain regions with variable effects on histamine release depending on the extent of wakefulness induction. In normal waking rats, eszopiclone only transiently suppressed histamine secretion, whereas this suppression was more obvious under caffeine-induced wakefulness. Compared with the GABA A modulator eszopiclone, DORA-22 elicits a neurotransmitter profile consistent with wake reduction that does not impinge on neurotransmitter levels associated with cognition and rapid eye movement sleep. © 2017 International Society for Neurochemistry.

  15. Novel immunotoxin: a fusion protein consisting of gelonin and an acetylcholine receptor fragment as a potential immunotherapeutic agent for the treatment of Myasthenia gravis.

    Science.gov (United States)

    Hossann, Martin; Li, Zhuoyu; Shi, Yawei; Kreilinger, Ulrike; Büttner, Jörn; Vogel, Pia D; Yuan, Jingming; Wise, John G; Trommer, Wolfgang E

    2006-03-01

    In continuation of our attempts for antigen-specific suppression of the immune system [I.L. Urbatsch, R.K.M. Sterz, K. Peper, W.E. Trommer, Eur. J. Immunol. 23(1993) 776-779] a novel fusion protein composed of amino acids 4-181 of the extracellular domain of the alpha-subunit of the human muscle acetylcholine receptor and the plant toxin gelonin was expressed in Escherichia coli. The fusion protein formed inclusion bodies but could be solubilized in the presence of guanidinium hydrochloride. After a simple two step purification and refolding procedure, it exhibited a native structure at least in the main immunogenic region as shown by antibodies recognizing a conformational epitope. Half maximal inhibition of translation was achieved at 46 ng/ml as compared to 4.6 ng/ml for native and 2.4 for recombinant gelonin. Its use as therapeutic agent for the treatment of Myasthenia gravis was investigated in an animal model. Female Lewis rats were immunized with complete acetylcholine receptor from the electric ray Torpedo californica and developed thereafter experimental autoimmune M. gravis. Quantitative assessment of the disease was achieved by repetitive stimulation of the Nervus tibialis. Rats showed no symptoms of M. gravis, neither visually nor electrophysiologically after treatment with the fusion protein as determined one and seven weeks after the second application. This approach may also be useful for the therapy of further autoimmune diseases by substituting other autoantigens for the AchR fragment in the fusion protein.

  16. Supplementing female rats with DHA-lysophosphatidylcholine increases docosahexaenoic acid and acetylcholine contents in the brain and improves the memory and learning capabilities of the pups.

    Directory of Open Access Journals (Sweden)

    Rojas, I.

    2010-03-01

    Full Text Available Docosahexaenoic acid (DHA is supplied to the foetus and newborn through the mother from their own reserves and their diet. No consensus about the best form to supplement DHA has been established. We propose that DHAcontaining lysophosphatidylcholine (DHA-LPC, obtained from DHA-rich eggs may be a suitable form of DHA and choline (the precursor of acetylcholine supplementation. We evaluated the effectiveness of DHA-LPC to increase DHA and acetylcholine concentration in the brain of pups born from female rats supplemented with DHA-LPC before and during pregnancy. We also evaluated the effect of DHA supplementation on learning and memory capabilities of pups through the Skinner test for operant conditioning. Female Wistar rats received 40-day supplementation of DHA-LPC (8 mg DHA/kg b.w/daily., before and during pregnancy. After delivery, plasma, erythrocyte, liver, and adipose tissue DHA and plasma choline were analyzed. Brains from 60 day-old pups separated into frontal cortex, cerebellum, striatum, hippocampus, and occipital cortex, were assessed for DHA, acetylcholine, and acetylcholine transferase (CAT activity. Pups were subjected to the Skinner box test. DHA-LPC supplementation produces higher choline and liver DHA contents in the mother’s plasma and increases the pups’ DHA and acetylcholine in the cerebellum and hippocampus. CAT was not modified by supplementation. The Skinner test shows that pups born from DHA-LPC supplemented mothers exhibit better scores of learning and memory than the controls. Conclusion: DHA-LPC may be an adequate form for DHA supplementation during the perinatal period.El ácido docosahexaenoico (DHA que requiere el feto y el recién nacido lo aporta la madre desde sus reservas y la dieta, por lo cual se sugiere suplementar a la madre con DHA. No hay consenso sobre la mejor forma de suplementación. Proponemos que un lisofosfolípido que contiene DHA y colina (DHA-LPC obtenido de huevos con alto contenido de DHA es

  17. Acetylcholinesterase and Acetylcholine Receptor.

    Science.gov (United States)

    1986-01-21

    Cl3CCH2OH) binds similarly to its carbon analogue, neopentyl alcohol, and chloral binds better than its carbon analogue, pivalaldehyde. In the latter case...III. Since the 3H-DFP was obtained in propylene glycol , the stability of DFP in the hydroxylic solvent and thus its true concentration was investi...solution of unlabeled DFP in propylene glycol was obtained from NEN for use in model experi- ments and found to have no inhibiting activity. We turned

  18. THE EFFECT OF INTRASTRIATAL APPLICATION OF DIRECTLY AND INDIRECTLY ACTING DOPAMINE AGONISTS AND ANTAGONISTS ON THE INVIVO RELEASE OF ACETYLCHOLINE MEASURED BY BRAIN MICRODIALYSIS - THE IMPORTANCE OF THE POSTSURGERY INTERVAL

    NARCIS (Netherlands)

    DEBOER, P; DAMSMA, G; SCHRAM, Q; STOOF, JC; ZAAGSMA, J; WESTERINK, BHC

    The effect of intrastriatal application of D-1, D-2 and indirect dopaminergic drugs on the release of striatal acetylcholine as a function of the post-implantation intervals was studied using in vivo microdialysis. The dopamine D-2 agonists LY 171555 and (-)N0437 inhibited the release of striatal

  19. Repeated administration of alpha7 nicotinic acetylcholine receptor (nAChR) agonists, but not positive allosteric modulators, increases alpha7 nAChR levels in the brain

    DEFF Research Database (Denmark)

    Christensen, Ditte Z; Mikkelsen, Jens D; Hansen, Henrik H

    2010-01-01

    The alpha7 nicotinic acetylcholine receptor (nAChR) is an important target for treatment of cognitive deficits in schizophrenia and Alzheimer's disease. However, the receptor desensitizes rapidly in vitro, which has led to concern regarding its applicability as a clinically relevant drug target...

  20. Fetal-muscle type nicotinic acetylcholine receptor activation in TE-671 cells, and inhibition of fetal movement in a day 40 pregnant goat model by optical isomers of the piperidine alkaloid coniine

    Science.gov (United States)

    Coniine is an optically active toxic piperidine alkaloid and nicotinic acetylcholine receptor (nAChR) agonist found in poison hemlock (Conium maculatum L.). Coniine teratogenicity is hypothesized to be due to the binding, activation, and prolonged desensitization of fetal muscle-type nAChR which re...