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Sample records for acetylcholine receptor subunits

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

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

    1998-01-01

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

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

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

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

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

    2000-01-01

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

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

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

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

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

    2004-04-01

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

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

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

    2001-01-01

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

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

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

    2009-01-01

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

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

    Emiliane Taillebois

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

  9. Distribution of the a2, a3, and a5 nicotinic acetylcholine receptor subunits in the chick brain

    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

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

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

    2009-01-01

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

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

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

    1986-03-05

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

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

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

    2011-01-01

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

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

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

    2014-01-01

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

  14. Tubular crystals of acetylcholine receptor

    1984-01-01

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

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

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

    2014-01-01

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

  16. Solution structure of α-conotoxin PIA, a novel antagonist of α6 subunit containing nicotinic acetylcholine receptors

    α-Conotoxin PIA is a novel nicotinic acetylcholine receptor (nAChR) antagonist isolated from Conus purpurascens that targets nAChR subtypes containing α6 and α3 subunits. α-conotoxin PIA displays 75-fold higher affinity for rat α6/α3β2β3 nAChRs than for rat α3β2 nAChRs. We have determined the three-dimensional structure of α-conotoxin PIA by nuclear magnetic resonance spectroscopy. The α-conotoxin PIA has an 'ω-shaped' overall topology as other α4/7 subfamily conotoxins. Yet, unlike other neuronally targeted α4/7-conotoxins, its N-terminal tail Arg1-Asp2-Pro3 protrudes out of its main molecular body because Asp2-Pro3-Cys4-Cys5 forms a stable type I β-turn. In addition, a kink introduced by Pro15 in the second loop of this toxin provides a distinct steric and electrostatic environment from those in α-conotoxins MII and GIC. By comparing the structure of α-conotoxin PIA with other functionally related α-conotoxins we suggest structural features in α-conotoxin PIA that may be associated with its unique receptor recognition profile

  17. The α6 nicotinic acetylcholine receptor subunit of Frankliniella occidentalis is not involved in resistance to spinosad.

    Hou, Wenjie; Liu, Qiulei; Tian, Lixia; Wu, Qingjun; Zhang, Youjun; Xie, Wen; Wang, Shaoli; Miguel, Keri San; Funderburk, Joe; Scott, Jeffrey G

    2014-05-01

    Insects evolve resistance which constrains the sustainable use of insecticides. Spinosyns, a class of environmentally-friendly macrolide insecticides, is not an exception. The mode of inheritance and the mechanisms of resistance to spinosad (the most common spinosyn insecticide) in Frankliniella occidentalis (Western flower thrips, WFT) were investigated in this study. Resistance (170,000-fold) was autosomal and completely recessive. Recent studies showed that deletion of the nicotinic acetylcholine receptor α6 subunit gene resulted in strains of Drosophila melanogaster, Plutella xylostella and Bactrocera dorsalis that are resistant to spinosad, indicating that nAChRα6 subunit maybe important for the toxic action of this insecticide. Conversely, a G275E mutation of this subunit in F. occidentalis was recently proposed as the mechanism of resistance to spinosad. We cloned and characterized nAChRα6 from three susceptible and two spinosad resistant strains from China and the USA. The Foα6 cDNA is 1873bp and the open reading frame is 1458bp which encodes 485 amino acid residues with a predicted molecular weight of 53.5-kDa, the 5' and 3' UTRs are 121 and 294bp, respectively. There was no difference in the cDNA sequence between the resistant and susceptible thrips, suggesting the G275E mutation does not confer resistance in these populations. Ten isoforms of Foα6, arising from alternative splicing, were isolated and did not differ between the spinosad-susceptible and resistant strains. Quantitative real time PCR analysis showed Foα6 was highly expressed in the first instar larva, pupa and adult, and the expression levels were 3.67, 2.47, 1.38 times that of the second instar larva. The expression level was not significantly different between the susceptible and resistant strains. These results indicate that Foα6 is not involved in resistance to spinosad in F. occidentalis from China and the USA. PMID:24861935

  18. Novel role for cyclin-dependent kinase 2 in neuregulin-induced acetylcholine receptor epsilon subunit expression in differentiated myotubes.

    Lu, Gang; Seta, Karen A; Millhorn, David E

    2005-06-10

    Cyclin-dependent kinases (CDKs) are a family of evolutionarily conserved serine/threonine kinases. CDK2 acts as a checkpoint for the G(1)/S transition in the cell cycle. Despite a down-regulation of CDK2 activity in postmitotic cells, many cell types, including muscle cells, maintain abundant levels of CDK2 protein. This led us to hypothesize that CDK2 may have a function in postmitotic cells. We show here for the first time that CDK2 can be activated by neuregulin (NRG) in differentiated C2C12 myotubes. In addition, this activity is required for expression of the acetylcholine receptor (AChR) epsilon subunit. The switch from the fetal AChRgamma subunit to the adult-type AChRepsilon is required for synapse maturation and the neuromuscular junction. Inhibition of CDK2 activity with either the specific CDK2 inhibitory peptide Tat-LFG or by RNA interference abolished neuregulin-induced AChRepsilon expression. Neuregulin-induced activation of CDK2 also depended on the ErbB receptor, MAPK, and PI3K, all of which have previously been shown to be required for AChRepsilon expression. Neuregulin regulated CDK2 activity through coordinating phosphorylation of CDK2 on Thr-160, accumulation of CDK2 in the nucleus, and down-regulation of the CDK2 inhibitory protein p27 in the nucleus. In addition, we also observed a novel mechanism of regulation of CDK2 activity by a low molecular weight variant of cyclin E in response to NRG. These findings establish CDK2 as an intermediate molecule that integrates NRG-activated signals from both the MAPK and PI3K pathways to AChRepsilon expression and reveal an undiscovered physiological role for CDK2 in postmitotic cells. PMID:15824106

  19. Direction-Specific Disruption of Subcortical Visual Behavior and Receptive Fields in Mice Lacking the Beta2 Subunit of Nicotinic Acetylcholine Receptor

    Wang, Lupeng; Rangarajan, Krsna V.; Lawhn-Heath, Courtney A.; Sarnaik, Rashmi; Wang, Bor-Shuen; Liu, Xiaorong; Cang, Jianhua

    2009-01-01

    Retinotopic mapping is a basic feature of visual system organization, but its role in processing visual information is unknown. Mutant mice lacking β2 subunit of nicotinic acetylcholine receptor have imprecise maps in both visual cortex (V1) and the superior colliculus (SC) due to the disruption of spontaneous retinal activity during development. Here, we use behavioral and physiological approaches to study their visual functions. We find that β2−/− mice fail to track visual stimuli moving al...

  20. Polymorphisms in the neural nicotinic acetylcholine receptor α4 subunit (CHRNA4) are associated with ADHD in a genetic isolate.

    Wallis, Deeann; Arcos-Burgos, Mauricio; Jain, Mahim; Castellanos, F Xavier; Palacio, Juan David; Pineda, David; Lopera, Francisco; Stanescu, Horia; Pineda, Daniel; Berg, Kate; Palacio, Luis Guillermo; Bailey-Wilson, Joan E; Muenke, Maximilian

    2009-05-01

    The neural nicotinic acetylcholine receptor α4 subunit (CHRNA4), at 20q13.2-q13.3, is an important candidate gene for conferring susceptibility to attention deficit/hyperactivity disorder (ADHD). Several studies have already looked for association/linkage between ADHD and CHRNA4 in different populations. We used the Pedigree Disequilibrium Test to search for evidence of association between ADHD and six SNP marker loci in families from the isolated Paisa population. We found that the T allele of SNP rs6090384 exhibits a deficit of transmission in unaffected individuals (OR = 5.43, IC 1.54-19.13) (global P value = 0.014). We also found significant association and linkage to extended haplotypes rs2273502-rs6090384 (combination of variants C-T, respectively) (P = 0.02) and rs6090384-rs6090387 (P = 0.04) (combination of variants T-G, respectively). SNP rs6090384, variant T, has also been reported to be associated with inattention in a previous study. This makes ours the ninth study to examine the association of CHRNA4 with ADHD and the seventh one to find evidence for association in a population with a different ethnicity. PMID:21432576

  1. Up-regulated expression of the alpha7 nicotinic acetylcholine receptor subunit on inflammatory infiltrates during Dictyocaulus viviparus infection.

    Lazari, O; Kipar, A; Johnson, D R; Selkirk, M E; Matthews, J B

    2006-09-01

    Cholinergic signalling is known to affect immune cell function, but few studies have addressed its relevance during nematode infection. We therefore analysed the anatomical distribution and expression pattern of the nicotinic acetylcholine receptor (nAChR) alpha7 subunit in lungs obtained from Dictyocaulus viviparus-infected and uninfected control cattle. The analysis was performed on trachea and lung parenchyma from uninfected animals and animals necropsied at 15, 22 and 43 days post-infection (DPI). Localization of the alpha7 nAChR was evaluated by immunohistology and mRNA expression analysed by gene-specific reverse transcription-polymerase chain reaction (RT-PCR). In uninfected animals, tracheal, bronchial and bronchiolar epithelium and smooth muscle cells constitutively expressed the alpha7 nAChR, as did type I and II alveolar epithelial cells and alveolar macrophages and a few infiltrating leucocytes. By 15 DPI, immunohistology revealed a massive influx of alpha7 nAChR+ inflammatory cells into the lung parenchyma and tracheal wall. This was reflected in the RT-PCR results. At later time points, both parenchyma and tracheal wall contained large numbers of alpha7 nAChR+ leucocytes, but detection of transcript was restricted to the trachea. Recruitment of nAChR-containing leucocytes to the lungs of D. viviparus-infected cattle suggests that these cells may represent possible downstream targets for parasite-secreted acetylcholinesterases. PMID:16916366

  2. Mutation of a nicotinic acetylcholine receptor β subunit is associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae

    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

  3. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta

  4. Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

    Machaalani, R., E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)

    2014-05-01

    Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

  5. Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR.

    Olena Filchakova

    Full Text Available Nicotinic acetylcholine receptors (nAChRs containing the α9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The α9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the α10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human α9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human α9α10 nAChRs is very low compared to that of rat α9α10 nAChRs. When oocytes were co-injected with cRNA of α9 and α10 subunits of human versus those of rat, oocytes with the rat α9 human α10 combination had an ∼-fold higher level of acetylcholine-gated currents (I(ACh than those with the human α9 rat α10 combination, suggesting difficulties with human α9 expression. When the ratio of injected human α9 cRNA to human α10 cRNA was increased from 1∶1 to 5∶1, I(ACh increased 36-fold (from 142±23 nA to 5171±748 nA. Functional expression of human α9-containing receptors in oocytes was markedly improved by appending the 5'-untranslated region of alfalfa mosaic virus RNA4 to the 5'-leader sequence of the α9 subunit cRNA. This increased the functional expression of homomeric human α9 receptors by 70-fold (from 7±1 nA to 475±158 nA and of human α9α10 heteromeric receptors by 80-fold (from 113±62 nA to 9192±1137 nA. These findings indicate the importance of the composition of the 5' untranslated leader sequence for expression of α9-containing nAChRs.

  6. Functional expression of human α9* nicotinic acetylcholine receptors in X. laevis oocytes is dependent on the α9 subunit 5' UTR.

    Filchakova, Olena; McIntosh, J Michael

    2013-01-01

    Nicotinic acetylcholine receptors (nAChRs) containing the α9 subunit are expressed in a wide variety of non-neuronal tissues ranging from immune cells to breast carcinomas. The α9 subunit is able to assemble into a functional homomeric nAChR and also co-assemble with the α10 subunit into functional heteromeric nAChRs. Despite the increasing awareness of the important roles of this subunit in vertebrates, the study of human α9-containing nAChRs has been severely limited by difficulties in its expression in heterologous systems. In Xenopus laevis oocytes, functional expression of human α9α10 nAChRs is very low compared to that of rat α9α10 nAChRs. When oocytes were co-injected with cRNA of α9 and α10 subunits of human versus those of rat, oocytes with the rat α9 human α10 combination had an ∼-fold higher level of acetylcholine-gated currents (I(ACh)) than those with the human α9 rat α10 combination, suggesting difficulties with human α9 expression. When the ratio of injected human α9 cRNA to human α10 cRNA was increased from 1∶1 to 5∶1, I(ACh) increased 36-fold (from 142±23 nA to 5171±748 nA). Functional expression of human α9-containing receptors in oocytes was markedly improved by appending the 5'-untranslated region of alfalfa mosaic virus RNA4 to the 5'-leader sequence of the α9 subunit cRNA. This increased the functional expression of homomeric human α9 receptors by 70-fold (from 7±1 nA to 475±158 nA) and of human α9α10 heteromeric receptors by 80-fold (from 113±62 nA to 9192±1137 nA). These findings indicate the importance of the composition of the 5' untranslated leader sequence for expression of α9-containing nAChRs. PMID:23717646

  7. The nicotinic acetylcholine receptors of the parasitic nematode Ascaris suum: formation of two distinct drug targets by varying the relative expression levels of two subunits.

    Sally M Williamson

    2009-07-01

    Full Text Available Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect approximately 1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5:1 (Asu-unc-38ratioAsu-unc-29, nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1:5 Asu-unc-38ratioAsu-unc-29, levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the

  8. A Novel α2/α4 Subtype-selective Positive Allosteric Modulator of Nicotinic Acetylcholine Receptors Acting from the C-tail of an α Subunit.

    Wang, Jingyi; Kuryatov, Alexander; Jin, Zhuang; Norleans, Jack; Kamenecka, Theodore M; Kenny, Paul J; Lindstrom, Jon

    2015-11-27

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChR) are important therapeutic candidates as well as valuable research tools. We identified a novel type II PAM, (R)-7-bromo-N-(piperidin-3-yl)benzo[b]thiophene-2-carboxamide (Br-PBTC), which both increases activation and reactivates desensitized nAChRs. This compound increases acetylcholine-evoked responses of α2* and α4* nAChRs but is without effect on α3* or α6* nAChRs (* indicates the presence of other nAChR subunits). Br-BPTC acts from the C-terminal extracellular sequences of α4 subunits, which is also a PAM site for steroid hormone estrogens such as 17β-estradiol. Br-PBTC is much more potent than estrogens. Like 17β-estradiol, the non-steroid Br-PBTC only requires one α4 subunit to potentiate nAChR function, and its potentiation is stronger with more α4 subunits. This feature enables Br-BPTC to potentiate activation of (α4β2)(α6β2)β3 but not (α6β2)2β3 nAChRs. Therefore, this compound is potentially useful in vivo for determining functions of different α6* nAChR subtypes. Besides activation, Br-BPTC affects desensitization of nAChRs induced by sustained exposure to agonists. After minutes of exposure to agonists, Br-PBTC reactivated short term desensitized nAChRs that have at least two α4 subunits but not those with only one. Three α4 subunits were required for Br-BPTC to reactivate long term desensitized nAChRs. These data suggest that higher PAM occupancy promotes channel opening more efficiently and overcomes short and long term desensitization. This C-terminal extracellular domain could be a target for developing subtype or state-selective drugs for nAChRs. PMID:26432642

  9. Individual response speed is modulated by variants of the gene encoding the alpha 4 sub-unit of the nicotinic acetylcholine receptor (CHRNA4).

    Schneider, Katja Kerstin; Schote, Andrea B; Meyer, Jobst; Markett, Sebastian; Reuter, Martin; Frings, Christian

    2015-05-01

    Acetylcholine (ACh) is a known modulator of several domains of cognition, among them attention, memory and learning. The neurotransmitter also influences the speed of information processing, particularly the detection of targets and the selection of suitable responses. We examined the effect of the rs1044396 (C/T) polymorphism of the gene encoding the nicotinic acetylcholine receptor α4-subunit (CHRNA4) on response speed and selective visual attention. To this end, we administered a Stroop task, a Negative priming task and an exogenous Posner-Cuing task to healthy participants (n = 157). We found that the CHRNA4 rs1044396 polymorphism modulated the average reaction times (RTs) across all three tasks. Dependent on the C allele dosage, the RTs linearly increased. Homozygous T allele carriers were always fastest, while homozygous C allele carriers were always slowest. We did not observe effects of this polymorphism on selective attention. In sum, we conclude that naturally occurring variations within the cholinergic system influence an important factor of information processing. This effect might possibly be produced by the neuromodulator system rather than the deterministic system of cortical ACh. PMID:25639542

  10. Immunisation with Torpedo acetylcholine receptor.

    Elfman, L

    1984-01-01

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

  11. Association of Common Polymorphisms in the Nicotinic Acetylcholine Receptor Alpha4 Subunit Gene with an Electrophysiological Endophenotype in a Large Population-Based Sample

    Mobascher, A.; Diaz-Lacava, A.; Wagner, M.; Gallinat, J.; Wienker, T. F.; Drichel, D.; Becker, T.; Steffens, M.; Dahmen, N.; Gründer, G.; Thürauf, N.; Kiefer, F.; Kornhuber, J.; Toliat, M. R.; Thiele, H.; Nürnberg, P.; Steinlein, O.; Winterer, G.

    2016-01-01

    Variation in genes coding for nicotinic acetylcholine receptor (nAChR) subunits affect cognitive processes and may contribute to the genetic architecture of neuropsychiatric disorders. Single nucleotide polymorphisms (SNPs) in the CHRNA4 gene that codes for the alpha4 subunit of alpha4/beta2-containing receptors have previously been implicated in aspects of (mostly visual) attention and smoking-related behavioral measures. Here we investigated the effects of six synonymous but functional CHRNA4 exon 5 SNPs on the N100 event-related potential (ERP), an electrophysiological endophenotype elicited by a standard auditory oddball. A total of N = 1,705 subjects randomly selected from the general population were studied with electroencephalography (EEG) as part of the German Multicenter Study on nicotine addiction. Two of the six variants, rs1044396 and neighboring rs1044397, were significantly associated with N100 amplitude. This effect was pronounced in females where we also observed an effect on reaction time. Sequencing of the complete exon 5 region in the population sample excluded the existence of additional/functional variants that may be responsible for the observed effects. This is the first large-scale population-based study investigation the effects of CHRNA4 SNPs on brain activity measures related to stimulus processing and attention. Our results provide further evidence that common synonymous CHRNA4 exon 5 SNPs affect cognitive processes and suggest that they also play a role in the auditory system. As N100 amplitude reduction is considered a schizophrenia-related endophenotype the SNPs studied here may also be associated with schizophrenia outcome measures. PMID:27054571

  12. A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.

    Zimmer, Christoph T; Garrood, William T; Puinean, A Mirel; Eckel-Zimmer, Manuela; Williamson, Martin S; Davies, T G Emyr; Bass, Chris

    2016-06-01

    Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target

  13. A family of acetylcholine-gated chloride channel subunits in Caenorhabditis elegans.

    Putrenko, Igor; Zakikhani, Mahvash; Dent, Joseph A

    2005-02-25

    The genome of the nematode Caenorhabditis elegans encodes a surprisingly large and diverse superfamily of genes encoding Cys loop ligand-gated ion channels. Here we report the first cloning, expression, and pharmacological characterization of members of a family of anion-selective acetylcholine receptor subunits. Two subunits, ACC-1 and ACC-2, form homomeric channels for which acetylcholine and arecoline, but not nicotine, are efficient agonists. These channels are blocked by d-tubocurarine but not by alpha-bungarotoxin. We provide evidence that two additional subunits, ACC-3 and ACC-4, interact with ACC-1 and ACC-2. The acetylcholine-binding domain of these channels appears to have diverged substantially from the acetylcholine-binding domain of nicotinic receptors. PMID:15579462

  14. An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

    Salmon, A M; Bruand, C; Cardona, A; Changeux, J P; Berrih-Aknin, S

    1998-06-01

    Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance. PMID:9616205

  15. Mutation (G275E) of the nicotinic acetylcholine receptor α6 subunit is associated with high levels of resistance to spinosyns in Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae).

    Silva, Wellington M; Berger, Madeleine; Bass, Chris; Williamson, Martin; Moura, Danielle M N; Ribeiro, Lílian M S; Siqueira, Herbert A A

    2016-07-01

    The tomato leafminer, Tuta absoluta, now a major pest of tomato crops worldwide, is primarily controlled using chemical insecticides. Recently, high levels of resistance to the insecticide spinosad have been described in T. absoluta populations in Brazil. Selection of a resistant field-collected strain led to very high levels of resistance to spinosad and cross-resistance to spinetoram, but not to other insecticides that target the nicotinic acetylcholine receptor (nAChR). In this study the mechanisms underlying resistance to spinosad were investigated using toxicological, biochemical and molecular approaches. Inhibition of metabolic enzymes using synergists and biochemical assessment of detoxification enzyme activity provided little evidence of metabolic resistance in the selected strain. Cloning and sequencing of the nAChR α6 subunit from T. absoluta, the spinosad target-site, from susceptible and spinosad-resistant strains were done to investigate the role of a target-site mechanism in resistance. A single nucleotide change was identified in exon 9 of the α6 subunit of the resistant strain, resulting in the replacement of the glycine (G) residue at position 275 observed in susceptible T. absoluta strains with a glutamic acid (E). A high-throughput DNA-based diagnostic assay was developed and used to assess the prevalence of the G275E mutation in 17 field populations collected from different geographical regions of Brazil. The resistant allele was found at low frequency, and in the heterozygous form, in seven of these populations but at much higher frequency and in the homozygous form in a population collected in the Iraquara municipality. The frequency of the mutation was significantly correlated with the mortality of these populations in discriminating dose bioassays. In summary our results provide evidence that the G275E mutation is an important mechanism of resistance to spinosyns in T. absoluta, and may be used as a marker for resistance monitoring in

  16. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits α7 and β2 in the sudden infant death syndrome (SIDS) brainstem

    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared α7 and β2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased α7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased β2 in the cNTS and increased β2 in the facial. When considering only the SIDS cohort: 1—cigarette smoke exposure was associated with increased α7 in the vestibular nucleus and increased β2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2—there was a gender interaction for α7 in the gracile and cuneate, and β2 in the cNTS and rostral arcuate nucleus, and 3—there was no effect of sleep position on α7, but prone sleep was associated with decreased β2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of α7 and β2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure (β2), gender (α7 and β2) and sleep position (β2) evident. -- Highlights: ► The ‘normal’ response to smoke exposure is decreased α7 and β2 in certain nuclei. ► SIDS infants have decreased α7 in cNTS, Grac and Cun. ► SIDS infants have decreased β2 in cNTS and increased β2 in facial. ► The NTS is more sensitive to both α7 and β2 regulation in SIDS. ► Smoke exposure

  17. Effects of cigarette smoke exposure on nicotinic acetylcholine receptor subunits {alpha}7 and {beta}2 in the sudden infant death syndrome (SIDS) brainstem

    Machaalani, Rita, E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Say, Meichien [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); Waters, Karen A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia)

    2011-12-15

    It is postulated that nicotine, as the main neurotoxic constituent of cigarette smoke, influences SIDS risk through effects on nicotinic acetylcholine receptors (nAChRs) in brainstem nuclei that control respiration and arousal. This study compared {alpha}7 and {beta}2 nAChR subunit expression in eight nuclei of the caudal and rostral medulla and seven nuclei of the pons between SIDS (n = 46) and non-SIDS infants (n = 14). Evaluation for associations with known SIDS risk factors included comparison according to whether infants had a history of exposure to cigarette smoke in the home, and stratification for sleep position and gender. Compared to non-SIDS infants, SIDS infants had significantly decreased {alpha}7 in the caudal nucleus of the solitary tract (cNTS), gracile and cuneate nuclei, with decreased {beta}2 in the cNTS and increased {beta}2 in the facial. When considering only the SIDS cohort: 1-cigarette smoke exposure was associated with increased {alpha}7 in the vestibular nucleus and increased {beta}2 in the rostral dorsal motor nucleus of the vagus, rNTS and Cuneate, 2-there was a gender interaction for {alpha}7 in the gracile and cuneate, and {beta}2 in the cNTS and rostral arcuate nucleus, and 3-there was no effect of sleep position on {alpha}7, but prone sleep was associated with decreased {beta}2 in three nuclei of the pons. In conclusion, SIDS infants demonstrate differences in expression of {alpha}7 and {beta}2 nAChRs within brainstem nuclei that control respiration and arousal, which is independent on prior history of cigarette smoke exposure, especially for the NTS, with additional differences for smoke exposure ({beta}2), gender ({alpha}7 and {beta}2) and sleep position ({beta}2) evident. -- Highlights: Black-Right-Pointing-Pointer The 'normal' response to smoke exposure is decreased {alpha}7 and {beta}2 in certain nuclei. Black-Right-Pointing-Pointer SIDS infants have decreased {alpha}7 in cNTS, Grac and Cun. Black

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

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

    2014-03-25

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

  19. An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

    Salmon, A M; Bruand, C; Cardona, A; Changeux, J P; Berrih-Aknin, S.

    1998-01-01

    Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subu...

  20. Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels

    De Souza Silva, M. A.; Dolga, Amalia; Pieri, I.; Marchetti, L.; Eisel, U. L. M.; Huston, J. P.; Dere, E.

    2006-01-01

    It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-D-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the n

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

    Villarroel, A.; Sakmann, B

    1992-01-01

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

  2. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Esam E. El-Fakahany

    2010-08-01

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

  3. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    Jakubík, Jan; El-Fakahany, E. E.

    2010-01-01

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

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

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

    2016-03-01

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

  5. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

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

    2010-09-01

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

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

    Carlos M. Carballosa

    2016-07-01

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

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

    Maelle Jospin

    2009-12-01

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

  8. The α7 nicotinic acetylcholine receptor complex

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

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

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

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

    1987-09-01

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

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

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

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

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

    2013-01-01

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

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

    Elise Courtot

    2015-12-01

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

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

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

    2015-01-01

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

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

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

    2016-04-01

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

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

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

    2009-01-01

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

  16. Nicotinic acetylcholine receptors mediate lung cancer growth

    PaulDGardner

    2013-09-01

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

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

    Gu, Y.

    1989-01-01

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

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

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

    1987-09-01

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

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

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

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

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

    2003-01-10

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

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

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

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

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

    1991-04-23

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

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

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

    2007-01-01

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

  4. Overexpression of ??3/??5/??4 nicotinic receptor subunits modifies impulsive-like behavior

    Vi??als, Xavier; Molas Casacuberta, Susanna, 1985-; Gallego, Xavier; Fern??ndez Montes, Rub??n D.; Robledo, Patr??cia, 1958-; Dierssen, Mara; Maldonado, Rafael

    2012-01-01

    Recent studies have revealed that sequence variants in genes encoding the ??3/??5/??4 nicotinic acetylcholine receptor subunits are associated with nicotine dependence. In this study, we evaluated two specific aspects of executive functioning related to drug addiction (impulsivity and working memory) in transgenic mice over expressing ??3/??5/??4 nicotinic receptor subunits. Impulsivity and working memory were evaluated in an operant delayed alternation task, where mice must inhibit respondin...

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

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

  6. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Meyyammai Swaminathan

    2014-06-01

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

  7. Functional partial agonism at cloned human muscarinic acetylcholine receptors

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

    1996-01-01

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

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

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

    2015-08-01

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

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

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

    1998-05-01

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

  10. Voltage-Jump Relaxation Kinetics for Wild-type and Chimeric β Subunits of Neuronal Nicotinic Receptors

    Figl, Antonio; Labarca, Cesar; Davidson, Norman; Lester, Henry A.; Cohen, Bruce N.

    1996-01-01

    We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric β4/β2 subunits with α3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type α3β4 receptors displayed five- to eightfold slower voltage-jump relaxations than did the wild-type α3β2 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amp...

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

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

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

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

    2012-01-01

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

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

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

    2009-01-01

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

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

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

    2015-01-01

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

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

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

    1997-07-01

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

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

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

    2016-06-01

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

  17. Localization of muscarinic acetylcholine receptor in plant guard cells

    2001-01-01

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

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

    Abagyan Ruben

    2002-01-01

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

  19. Cocaine inhibition of nicotinic acetylcholine receptors influences dopamine release

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

    2014-01-01

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

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

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

    2011-01-01

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

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

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

  2. The duplicated α7 subunits assemble and form functional nicotinic receptors with the full-length α7

    Wang, Ying; Xiao, Cheng; Indersmitten, Tim; Freedman, Robert; Leonard, Sherry; Lester, Henry A.

    2014-01-01

    The α7 nicotinic acetylcholine receptor gene (CHRNA7) is linked to schizophrenia. A partial duplication of CHRNA7 (CHRFAM7A) is found in humans on 15q13-14. Exon 6 of CHRFAM7A harbors a 2 base pair deletion polymorphism, CHRFAM7AΔ2bp, which is also associated with schizophrenia. To understand the effects of the duplicated subunits on α7 receptors, we fused α7, dupα7, and dupΔα7 subunits with various fluorescent proteins. The duplicated subunits co-localized with full-length α7 subunits in mou...

  3. A new family of insect muscarinic acetylcholine receptors.

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

    2016-08-01

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

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

    Chur Chin

    2010-04-01

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

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

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

    2013-07-01

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

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

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

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

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

    2016-01-01

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

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

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

    2016-01-01

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

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

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

    2011-01-01

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

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

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

    2009-01-01

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

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

    Qiang LIU; Jie WU

    2006-01-01

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

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

    Jian-xin SHEN; Jerrel L YAKEL

    2009-01-01

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

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

    Zhang Chuan-Xi

    2007-09-01

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

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

    Shuyin Li; Liming Zou; Carry Pope

    2008-01-01

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

  15. Schizophrenia and the alpha7 nicotinic acetylcholine receptor.

    Martin, Laura F; Freedman, Robert

    2007-01-01

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

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

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

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

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

    2013-01-01

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

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

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

    2015-01-01

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

  19. Regulation of Glutamate Receptors by Their Auxiliary Subunits

    Tomita, Susumu

    2010-01-01

    Glutamate receptors are major excitatory receptors in the brain. Recent findings have established auxiliary subunits of glutamate receptors as critical modulators of synaptic transmission, synaptic plasticity and neurological disorder. The elucidation of the molecular rules governing glutamate receptors and subunits will improve our understanding of synapses and of neural-circuit regulation in the brain.

  20. Multiple transmembrane binding sites for p-trifluoromethyldiazirinyl-etomidate, a photoreactive Torpedo nicotinic acetylcholine receptor allosteric inhibitor.

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

    2011-06-10

    Photoreactive derivatives of the general anesthetic etomidate have been developed to identify their binding sites in γ-aminobutyric acid, type A and nicotinic acetylcholine receptors. One such drug, [(3)H]TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl-[(3)H]1-(1-phenylethyl)-1H-imidazole-5-carboxylate), acts as a positive allosteric potentiator of Torpedo nACh receptor (nAChR) and binds to a novel site in the transmembrane domain at the γ-α subunit interface. To extend our understanding of the locations of allosteric modulator binding sites in the nAChR, we now characterize the interactions of a second aryl diazirine etomidate derivative, TFD-etomidate (ethyl-1-(1-(4-(3-trifluoromethyl)-3H-diazirin-3-yl)phenylethyl)-1H-imidazole-5-carboxylate). TFD-etomidate inhibited acetylcholine-induced currents with an IC(50) = 4 μM, whereas it inhibited the binding of [(3)H]phencyclidine to the Torpedo nAChR ion channel in the resting and desensitized states with IC(50) values of 2.5 and 0.7 mm, respectively. Similar to [(3)H]TDBzl-etomidate, [(3)H]TFD-etomidate bound to a site at the γ-α subunit interface, photolabeling αM2-10 (αSer-252) and γMet-295 and γMet-299 within γM3, and to a site in the ion channel, photolabeling amino acids within each subunit M2 helix that line the lumen of the ion channel. In addition, [(3)H]TFD-etomidate photolabeled in an agonist-dependent manner amino acids within the δ subunit M2-M3 loop (δIle-288) and the δ subunit transmembrane helix bundle (δPhe-232 and δCys-236 within δM1). The fact that TFD-etomidate does not compete with ion channel blockers at concentrations that inhibit acetylcholine responses indicates that binding to sites at the γ-α subunit interface and/or within δ subunit helix bundle mediates the TFD-etomidate inhibitory effect. These results also suggest that the γ-α subunit interface is a binding site for Torpedo nAChR negative allosteric modulators (TFD-etomidate) and for positive

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

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

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

    1984-01-01

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

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

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

    2012-08-01

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

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

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

  5. Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine

    The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine ([125I]TID) was used to label acetylcholine receptor rich membranes purified from Torpedo californica electric organ. All four subunits of the acetylcholine receptor (AChR) were found to incorporate label, with the γ-subunit incorporating approximately 4 times as much as each of the other subunits. Carbamylcholine, an agonist, and histrionicotoxin, a noncompetitive antagonist, both strongly inhibited labeling of all AChR subunits in a specific and dose-dependent manner. In contrast, the competitive antagonist α-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effect on [125I]TID labeling of the AChR. The regions of the AChR α-subunit that incorporate [125]TID were mapped by Staphylococcus aureus V8 protest digestion. The carbamylcholine-sensitive site of labeling was localized to a 20-kDa V8 cleavage fragment that begins at Ser-173 and is of sufficient length to contain the three hydrophobic regions M1, M2, and M3. A 10-kDa fragment beginning at Asn-339 and containing the hydrophobic region M4 also incorporated [125I]TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the α-subunit amino terminus, incorporated no detectable [125I]TID. The mapping results place constraints on suggested models of AChR subunit topology

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

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

  7. Effects of two oxadiazolidinones on cholinesterases and acetylcholine receptors

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

  8. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

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

    2015-10-01

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

  9. Determinants of zinc potentiation on the alpha4 subunit of neuronal nicotinic receptors.

    Hsiao, Bernard; Mihalak, Karla B; Repicky, Sarah E; Everhart, Drew; Mederos, Ana H; Malhotra, Arun; Luetje, Charles W

    2006-01-01

    We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the alpha4beta2 and alpha4beta4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat alpha4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate. Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that alpha4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas alpha4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of alpha4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepine-binding site on GABA(A) receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA(A) receptors. PMID:16189299

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

    Schuller Hildegard M; Dhar Madhu; Plummer Howard K

    2005-01-01

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

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

    AndrewR.Tapper

    2013-04-01

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

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

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

    1992-01-01

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

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

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

    1992-01-01

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

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

    Zuo Jun Ren

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

  15. Cocaine Inhibition of Nicotinic Acetylcholine ReceptorsInfluences Dopamine Release

    Alexandra eAcevedo-Rodriguez

    2014-09-01

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

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

    Layla AZAM; J Michael MCINTOSH

    2009-01-01

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

  17. Multiple Nicotinic Acetylcholine Receptor Subtypes in the Mouse Amygdala Regulate Affective Behaviors and Response to Social Stress.

    Mineur, Yann S; Fote, Gianna M; Blakeman, Sam; Cahuzac, Emma L M; Newbold, Sylvia A; Picciotto, Marina R

    2016-05-01

    Electrophysiological and neurochemical studies implicate cholinergic signaling in the basolateral amygdala (BLA) in behaviors related to stress. Both animal studies and human clinical trials suggest that drugs that alter nicotinic acetylcholine receptor (nAChR) activity can affect behaviors related to mood and anxiety. Clinical studies also suggest that abnormalities in cholinergic signaling are associated with major depressive disorder, whereas pre-clinical studies have implicated both β2 subunit-containing (β2*) and α7 nAChRs in the effects of nicotine in models of anxiety- and depression-like behaviors. We therefore investigated whether nAChR signaling in the amygdala contributes to stress-mediated behaviors in mice. Local infusion of the non-competitive non-selective nAChR antagonist mecamylamine or viral-mediated downregulation of the β2 or α7 nAChR subunit in the amygdala all induced robust anxiolytic- and antidepressant-like effects in several mouse behavioral models. Further, whereas α7 nAChR subunit knockdown was somewhat more effective at decreasing anxiety-like behavior, only β2 subunit knockdown decreased resilience to social defeat stress and c-fos immunoreactivity in the BLA. In contrast, α7, but not β2, subunit knockdown effectively reversed the effect of increased ACh signaling in a mouse model of depression. These results suggest that signaling through β2* nAChRs is essential for baseline excitability of the BLA, and a decrease in signaling through β2 nAChRs alters anxiety- and depression-like behaviors even in unstressed animals. In contrast, stimulation of α7 nAChRs by acetylcholine may mediate the increased depression-like behaviors observed during the hypercholinergic state observed in depressed individuals. PMID:26471256

  18. Mutations in GABAA receptor subunits associated with genetic epilepsies.

    Macdonald, Robert L; Kang, Jing-Qiong; Gallagher, Martin J

    2010-06-01

    Mutations in inhibitory GABAA receptor subunit genes (GABRA1, GABRB3, GABRG2 and GABRD) have been associated with genetic epilepsy syndromes including childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME), pure febrile seizures (FS), generalized epilepsy with febrile seizures plus (GEFS+), and Dravet syndrome (DS)/severe myoclonic epilepsy in infancy (SMEI). These mutations are found in both translated and untranslated gene regions and have been shown to affect the GABAA receptors by altering receptor function and/or by impairing receptor biogenesis by multiple mechanisms including reducing subunit mRNA transcription or stability, impairing subunit folding, stability, or oligomerization and by inhibiting receptor trafficking. PMID:20308251

  19. Nicotinic acetylcholine receptors: a comparison of the nAChRs of Caenorhabditis elegans and parasitic nematodes.

    Holden-Dye, Lindy; Joyner, Michelle; O'Connor, Vincent; Walker, Robert J

    2013-12-01

    Nicotinic acetylcholine receptors (nAChRs) play a key role in the normal physiology of nematodes and provide an established target site for anthelmintics. The free-living nematode, Caenorhabditis elegans, has a large number of nAChR subunit genes in its genome and so provides an experimental model for testing novel anthelmintics which act at these sites. However, many parasitic nematodes lack specific genes present in C. elegans, and so care is required in extrapolating from studies using C. elegans to the situation in other nematodes. In this review the properties of C. elegans nAChRs are reviewed and compared to those of parasitic nematodes. This forms the basis for a discussion of the possible subunit composition of nAChRs from different species of parasitic nematodes. Currently our knowledge on this is largely based on studies using heterologous expression and pharmacological analysis of receptor subunits in Xenopus laevis oocytes. It is concluded that more information is required regarding the subunit composition and pharmacology of endogenous nAChRs in parasitic nematodes. PMID:23500392

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

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

    2016-08-01

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

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

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

    2016-01-01

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

  2. Subunit structure of the follitropin receptor

    Both of the α and β subunits of intact human follitropin (FSH) were radioiodinated with 125I-FSH-sodium iodide and chloramine-T, and could be resolved on polyacrylamide gels (SDS-PAGE). The electrophoretic mobility of radioiodinated FSH α and β subunits as well as the αβ dimer changed markedly depending on the concentration of reducing agents. 125I-FSH (Ka = 1.4 x 1010 M-1), complexes to the receptor on procine granulosa cells or in Triton X-100 extracts, was affinity-crosslinked with a cleavable (nondisulfide) homobifunctional reagent, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, solubilized in sodium dodecyl sulfate with or without reducing agents, and electrophoresed. Crosslinked samples revealed three additional bands of slower electrophoretic mobility, corresponding to 65 (unreduced 62), 83 (unreduced 76) and 117 (unreduced 110)kDa, in addition to hormone bands. Formation of the three bands requires the 125I-FSH hormone to bind specifically to the receptor with subsequent cross-linking. The rate of formation and cleavage of the cross-linked complexes indicated a sequential and incremental addition of 22, 18, and 34 kDa components to the FSH αβ dimer. The results of reduction of cross-linked complexes demonstrated the existence of disulfide linkage between the three components. FSH was photoactively derivatized with N-hydroxysuccinimide ester of 4-azidobenzolyl-glycine and radioiodinated for photoaffinity labeling. When derivatized 125I-FSH (Ka = 1.12 1010 M-1) bound to the cell was photolyzed for cross-linking and resolved on the SDS-PAGE, two new bands (106 and 61 kDa) under reducing condition appeared in addition to the hormone bands. Upon reduction with dithiotheitol and second-dimensional electrophoresis, the unreduced 104 kDa (reduced 106 kDa) band released two small components 31 and 14 kDa

  3. Acetylcholine receptors in dementia and mild cognitive impairment

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

    2008-03-15

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

  4. Acetylcholine receptors in dementia and mild cognitive impairment

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

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

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

    2015-01-01

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

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

    L.F.S. Sampaio

    2005-04-01

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

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

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

    1992-01-01

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

  8. Increased Sensitivity of the Neuronal Nicotinic Receptor α2 Subunit Causes Familial Epilepsy with Nocturnal Wandering and Ictal Fear

    Aridon, Paolo; Marini, Carla; Di Resta, Chiara; Brilli, Elisa; De Fusco, Maurizio; Politi, Fausta; Parrini, Elena; Manfredi, Irene; Pisano, Tiziana; Pruna, Dario; Curia, Giulia; Cianchetti, Carlo; Pasqualetti, Massimo; Becchetti, Andrea; Guerrini, Renzo; Casari, Giorgio

    2006-01-01

    Sleep has traditionally been recognized as a precipitating factor for some forms of epilepsy, although differential diagnosis between some seizure types and parasomnias may be difficult. Autosomal dominant frontal lobe epilepsy is characterized by nocturnal seizures with hyperkinetic automatisms and poorly organized stereotyped movements and has been associated with mutations of the α4 and β2 subunits of the neuronal nicotinic acetylcholine receptor. We performed a clinical and molecular genetic study of a large pedigree segregating sleep-related epilepsy in which seizures are associated with fear sensation, tongue movements, and nocturnal wandering, closely resembling nightmares and sleep walking. We identified a new genetic locus for familial sleep-related focal epilepsy on chromosome 8p12.3-8q12.3. By sequencing the positional candidate neuronal cholinergic receptor α2 subunit gene (CHRNA2), we detected a heterozygous missense mutation, I279N, in the first transmembrane domain that is crucial for receptor function. Whole-cell recordings of transiently transfected HEK293 cells expressing either the mutant or the wild-type receptor showed that the new CHRNA2 mutation markedly increases the receptor sensitivity to acetylcholine, therefore indicating that the nicotinic α2 subunit alteration is the underlying cause. CHRNA2 is the third neuronal cholinergic receptor gene to be associated with familial sleep-related epilepsies. Compared with the CHRNA4 and CHRNB2 mutations reported elsewhere, CHRNA2 mutations cause a more complex and finalized ictal behavior. PMID:16826524

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

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

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

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

    2012-01-06

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

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

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

    2016-01-01

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

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

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

    2008-01-01

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

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

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

    2012-01-01

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

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

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

    2011-07-27

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

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

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

    2012-01-01

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

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

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

    1996-01-01

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

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

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

    1997-01-01

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

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

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

    2013-11-01

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

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

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

    1985-08-12

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

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

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

    2012-08-01

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

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

    MENG Fanxia; MIAO Long; ZHANG Shuqiu; LOU Chenghou

    2004-01-01

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

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

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

    1993-01-01

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

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

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

    2014-01-01

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

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

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

    2015-01-01

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

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

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

    2010-01-01

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

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

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

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

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

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

    1987-12-01

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

  8. A7DB: a relational database for mutational, physiological and pharmacological data related to the α7 nicotinic acetylcholine receptor

    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.

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

    Rubin, L L

    1985-01-01

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

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

    Jakubík, Jan; Randáková, Alena; Doležal, Vladimír

    2013-01-01

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

  11. CaMKIIα, a modulator of M4 muscarinic acetylcholine receptors

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

    2010-01-01

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

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

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

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

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

    2013-01-01

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

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

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

    2015-11-01

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

  15. Effects of mutations of a glutamine residue in loop D of the α7 nicotinic acetylcholine receptor on agonist profiles for neonicotinoid insecticides and related ligands

    Shimomura, Masaru; Okuda, Hiroshi; Matsuda, Kazuhiko; Komai, Koichiro; Akamatsu, Miki; Sattelle, David B

    2002-01-01

    Neonicotinoid insecticides are agonists of insect nicotinic acetylcholine receptors (AChRs) and show selective toxicity for insects over vertebrates. To elucidate the molecular basis of the selectivity, amino acid residues influencing neonicotinoid sensitivity were investigated by site-directed mutagenesis of the chicken α7 nicotinic AChR subunit, based on the crystal structure of an ACh binding protein (AChBP).In the ligand binding site of AChBP, Q55 in loop D is close to Y164 in loop F that...

  16. A spinosyn-sensitive Drosophila melanogaster nicotinic acetylcholine receptor identified through chemically induced target site resistance, resistance gene identification, and heterologous expression.

    Watson, Gerald B; Chouinard, Scott W; Cook, Kevin R; Geng, Chaoxian; Gifford, Jim M; Gustafson, Gary D; Hasler, James M; Larrinua, Ignacio M; Letherer, Ted J; Mitchell, Jon C; Pak, William L; Salgado, Vincent L; Sparks, Thomas C; Stilwell, Geoff E

    2010-05-01

    Strains of Drosophila melanogaster with resistance to the insecticides spinosyn A, spinosad, and spinetoram were produced by chemical mutagenesis. These spinosyn-resistant strains were not cross-resistant to other insecticides. The two strains that were initially characterized were subsequently found to have mutations in the gene encoding the nicotinic acetylcholine receptor (nAChR) subunit Dalpha6. Subsequently, additional spinosyn-resistant alleles were generated by chemical mutagenesis and were also found to have mutations in the gene encoding Dalpha6, providing convincing evidence that Dalpha6 is a target site for the spinosyns in D. melanogaster. Although a spinosyn-sensitive receptor could not be generated in Xenopus laevis oocytes simply by expressing Dalpha6 alone, co-expression of Dalpha6 with an additional nAChR subunit, Dalpha5, and the chaperone protein ric-3 resulted in an acetylcholine- and spinosyn-sensitive receptor with the pharmacological properties anticipated for a native nAChR. PMID:19944756

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

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

    2016-03-17

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

  18. Mutant GABA(A) receptor subunits in genetic (idiopathic) epilepsy.

    Hirose, Shinichi

    2014-01-01

    The γ-aminobutyric acid receptor type A (GABAA receptor) is a ligand-gated chloride channel that mediates major inhibitory functions in the central nervous system. GABAA receptors function mainly as pentamers containing α, β, and either γ or δ subunits. A number of antiepileptic drugs have agonistic effects on GABAA receptors. Hence, dysfunctions of GABAA receptors have been postulated to play important roles in the etiology of epilepsy. In fact, mutations or genetic variations of the genes encoding the α1, α6, β2, β3, γ2, or δ subunits (GABRA1, GABRA6, GABRB2, GABRB3, GABRG2, and GABRD, respectively) have been associated with human epilepsy, both with and without febrile seizures. Epilepsy resulting from mutations is commonly one of following, genetic (idiopathic) generalized epilepsy (e.g., juvenile myoclonic epilepsy), childhood absence epilepsy, genetic epilepsy with febrile seizures, or Dravet syndrome. Recently, mutations of GABRA1, GABRB2, and GABRB3 were associated with infantile spasms and Lennox-Gastaut syndrome. These mutations compromise hyperpolarization through GABAA receptors, which is believed to cause seizures. Interestingly, most of the insufficiencies are not caused by receptor gating abnormalities, but by complex mechanisms, including endoplasmic reticulum (ER)-associated degradation, nonsense-mediated mRNA decay, intracellular trafficking defects, and ER stress. Thus, GABAA receptor subunit mutations are now thought to participate in the pathomechanisms of epilepsy, and an improved understanding of these mutations should facilitate our understanding of epilepsy and the development of new therapies. PMID:25194483

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

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

    1980-01-01

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

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

    Schuller Hildegard M

    2005-04-01

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

  1. Molecular docking study on the α3β2 neuronal nicotinic acetylcholine receptor complexed with α-Conotoxin GIC

    Chewook Lee

    2012-05-01

    Full Text Available Nicotinic acetylcholine receptors (nAChRs are a diverse familyof homo- or heteropentameric ligand-gated ion channels.Understanding the physiological role of each nAChR subtypeand the key residues responsible for normal and pathologicalstates is important. α-Conotoxin neuropeptides are highly selectiveprobes capable of discriminating different subtypes ofnAChRs. In this study, we performed homology modeling togenerate the neuronal α3, β2 and β4 subunits using the x-raystructure of the α1 subunit as a template. The structures of theextracellular domains containing ligand binding sites in theα3β2 and α3β4 nAChR subtypes were constructed using MDsimulations and ligand docking processes in their free and ligand-bound states using α-conotoxin GIC, which exhibited thehighest α3β2 vs. α3β4 discrimination ratio. The results providea reasonable structural basis for such a discriminatoryability, supporting the idea that the present strategy can beused for future investigations on nAChR-ligand complexes.[BMB reports 2012; 45(5: 275-280

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

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

    2006-01-01

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

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

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

    2015-01-01

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

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

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

    2013-01-01

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

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

    Tomizawa, Motohiro; Casida, John E.

    1999-01-01

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

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

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

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

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

    1984-01-01

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

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

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

    2011-01-01

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

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

    1984-01-01

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

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

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

    2007-01-01

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

  11. Partial agonists and subunit selectivity at NMDA receptors

    Risgaard, Rune; Hansen, Kasper Bø; Clausen, Rasmus Prætorius

    2010-01-01

    Subunit-selective ligands for glutamate receptors remains an area of interest as glutamate is the major excitatory neurotransmitter in the brain and involved in a number of diseased states in the central nervous system (CNS). Few subtype-selective ligands are known, especially among the N...

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

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

    1993-01-01

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

  13. Role of β4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice.

    Harrington, Lauriane; Viñals, Xavier; Herrera-Solís, Andrea; Flores, Africa; Morel, Carole; Tolu, Stefania; Faure, Philippe; Maldonado, Rafael; Maskos, Uwe; Robledo, Patricia

    2016-06-01

    Nicotine exerts its psychopharmacological effects by activating the nicotinic acetylcholine receptor (nAChR), composed of alpha and/or beta subunits, giving rise to a diverse population of receptors with a distinct pharmacology. β4-containing (β4*) nAChRs are located almost exclusively in the habenulo-interpeduncular pathway. We examined the role of β4* nAChRs in the medial habenula (MHb) and the interpeduncular nucleus (IPN) in nicotine reinforcement using behavioral, electrophysiological, and molecular techniques in transgenic mice. Nicotine intravenous self-administration (IVSA) was lower in constitutive β4 knockout (KO) mice at all doses tested (7.5, 15, 30, and 60 μg/kg/infusion) compared with wild-type (WT) mice. In vivo microdialysis showed that β4KO mice have higher extracellular dopamine (DA) levels in the nucleus accumbens than in WT mice, and exhibit a differential sensitivity to nicotine-induced DA outflow. Furthermore, electrophysiological recordings in the ventral tegmental area (VTA) demonstrated that DA neurons of β4KO mice are more sensitive to lower doses of nicotine than that of WT mice. Re-expression of β4* nAChRs in IPN neurons fully restored nicotine IVSA, and attenuated the increased sensitivity of VTA DA neurons to nicotine. These findings suggest that β4* nAChRs in the IPN have a role in maintaining nicotine IVSA. PMID:26585290

  14. Accumulation of human full-length tau induces degradation of nicotinic acetylcholine receptor α4 via activating calpain-2

    Yin, Yaling; Wang, Yali; Gao, Di; Ye, Jinwang; Wang, Xin; Fang, Lin; Wu, Dongqin; Pi, Guilin; Lu, Chengbiao; Zhou, Xin-Wen; Yang, Ying; Wang, Jian-Zhi

    2016-01-01

    Cholinergic impairments and tau accumulation are hallmark pathologies in sporadic Alzheimer’s disease (AD), however, the intrinsic link between tau accumulation and cholinergic deficits is missing. Here, we found that overexpression of human wild-type full-length tau (termed hTau) induced a significant reduction of α4 subunit of nicotinic acetylcholine receptors (nAChRs) with an increased cleavage of the receptor producing a ~55kDa fragment in primary hippocampal neurons and in the rat brains, meanwhile, the α4 nAChR currents decreased. Further studies demonstrated that calpains, including calpain-1 and calpain-2, were remarkably activated with no change of caspase-3, while simultaneous suppression of calpain-2 by selective calpain-2 inhibitor but not calpain-1 attenuated the hTau-induced degradation of α4 nAChR. Finally, we demonstrated that hTau accumulation increased the basal intracellular calcium level in primary hippocampal neurons. We conclude that the hTau accumulation inhibits nAChRs α4 by activating calpain-2. To our best knowledge, this is the first evidence showing that the intracellular accumulation of tau causes cholinergic impairments. PMID:27277673

  15. Evaluation of the Nicotinic Acetylcholine Receptor-Associated Proteome at Baseline and Following Nicotine Exposure in Human and Mouse Cortex

    Esterlis, Irina; Stone, Kathryn L.; Grady, Sharon R.; Lindstrom, Jon M.; Marks, Michael J.

    2016-01-01

    Abstract Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein–protein interactions of high-affinity nAChRs containing the β2 subunit (β2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the β2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.

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

    Alexander V Chibalin

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

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

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

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

    Proska, J; Tucek, S

    1996-06-01

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

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

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

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

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

    2010-07-13

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

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

    Abbas, Muzaffar; Rahman, Shafiqur

    2016-07-15

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

  2. The α4β2 nicotinic acetylcholine receptor modulates autism-like behavioral and motor abnormalities in pentylenetetrazol-kindled mice.

    Takechi, Kenshi; Suemaru, Katsuya; Kiyoi, Takeshi; Tanaka, Akihiro; Araki, Hiroaki

    2016-03-15

    Epilepsy is associated with several psychiatric disorders, including cognitive impairment, autism and attention deficit/hyperactivity disorder (ADHD). However, the psychopathology of epilepsy is frequently unrecognized and untreated in patients. In the present study, we investigated the effects of ABT-418, a neuronal nicotinic acetylcholine receptor agonist, on pentylenetetrazol (PTZ)-kindled mice with behavioral and motor abnormalities. PTZ-kindled mice displayed impaired motor coordination (in the rotarod test), anxiety (in the elevated plus maze test) and social approach impairment (in the three-chamber social test) compared with control mice. ABT-418 treatment (0.05mg/kg, intraperitoneally) alleviated these behavioral abnormalities in PTZ-kindled mice. Immunolabeling of tissue sections demonstrated that expression of the α4 nicotinic acetylcholine receptor subunit in the medial habenula was similar in control and PTZ-kindled mice. However, expression was significantly decreased in the piriform cortex in PTZ-kindled mice. In addition, we examined the expression of the synaptic adhesion molecule neuroligin 3 (NLG3). NLG3 expression in the piriform cortex was significantly higher in PTZ-kindled mice compared with control mice. Collectively, our findings suggest that ADHD-like or autistic-like behavioral abnormalities associated with epilepsy are closely related to the downregulation of the α4 nicotinic receptor and the upregulation of NLG3 in the piriform cortex. In summary, this study indicates that ABT-418 might have therapeutic potential for attentional impairment in epileptic patients with psychiatric disorders such as autism and ADHD. PMID:26868186

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

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

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

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

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

    2012-01-01

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

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

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

    2010-01-01

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

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

    Crittenden Elizabeth L

    2004-07-01

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

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

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

  8. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stress can increase susceptibility to developing psychiatric disorders, including depression. Understanding the neurobiological mechanisms underlying stress resilience and susceptibility is key to identifying novel targets for the development of more effective treatments for stress-related psychiatric disorders. Here we show that specific isoforms of GABAB receptor subunits differentially regulate stress resilience. Specifically, GABAB(1a)−/− mice are more susceptible whereas GABAB(1b)−/− mic...

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

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

    2011-01-01

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

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

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

    2011-01-01

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

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

    McGranahan, Tresa Michelle

    2011-01-01

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

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

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

    2010-01-01

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

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

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

    2010-01-01

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

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

    1995-01-01

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

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

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

    2007-01-01

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

  16. Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction.

    Perry, Trent; Somers, Jason; Yang, Ying Ting; Batterham, Philip

    2015-09-01

    Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and

  17. Natural CD4~+CD25~+ regulatory T cells express α7-nicotinic acetylcholine receptor subunits%小鼠天然CD4~+CD25~+调节性T细胞表达α7烟碱型乙酰胆碱受体

    王大伟; 周荣斌; 姚咏明

    2010-01-01

    Objective To investigate whether CD4~+ CD25~+ regulatory T cells (Treg) from C57BL/6J mice express alpha7 nicotinic acetylcholine receptor (α7nAChR). Methods CD4~+ CD25~+ regulatory T cells were isolated from mouse splenocytes with a CD4~+ CD25~+ regulatory T Cell isolation kit (Mihenyi Bio-tee). The purity of isolated Tregs was analyzed by flow eytometry. Expressions of α7nAChR in mouse CD4~+ CD25~+ Tregs were examined by immunofluorescence staining, Western blotting, and reverse transeription-PCR, respectively. Results It was revealed by flow cytometry that Tregs could bind alpha-bungarotoxin (α-BGT)-F/TC, a specific α7 nAChR antagonist. Moreover, a positive binding to α-Bgt was also observed on the cell surface of Treg, as viewed by fluorescent confoeal microscopy. In addition, a clear band of a7nAChR with a molecular mass of approximately 55 kD was found from Tregs by Western blotting analysis, and α7nAChR mRNA was expressed with the expected size of 199 bp from Tregs by reverse transcription-PCR. Conclusion Natural CD4~+ CD25~+ Tregs from mice express α7nAChR.%目的 探讨C57BL/6J小鼠的天然CD4~+CD25~+调节性T细胞(CD4~+CD25~+Treg)是否存在α7烟碱型乙酰胆碱受体(a7nAchR).方法 使用小鼠调节性T细胞试剂盒分离小鼠脾脏CD4~+CD25~+Treg,流式细胞术鉴定CD4~+CD25~+Treg的纯度.分别采用免疫荧光染色、共聚焦湿微镜、Western印迹和逆转录聚合酶链反应检测Treg表面α7nAchR蛋白/基因表达.结果 α-银环蛇毒素-FITC染色、流式检测显示Treg细胞表面结合α-银环蛇毒素-FITC;共聚焦显微镜成像观察到Treg细胞表面结合大量α-银环蛇毒素;Western印迹检测证实Treg细胞样本中检测到了清楚的α7nAchR条带,分子量大小约为55 kD;RT-PCR分析发现Treg细胞样本中检测到了199 bp大小的特异性α7nAchR目的 基因条带.结论小鼠天然CD4~+CD25~+Treg细胞表达α7nAChR.

  18. Purification and subunit composition of atrial natriuretic peptide receptor

    A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4.0 X 10(-10)M. Upon treatment with 10 mM dithiothreitol, the purified receptor migrated as a single band at Mr 60,500 in NaDodSO4/polyacrylamide gel electrophoresis. These findings show that the holoreceptor for ANP in vascular tissue is composed of two subunits of identical apparent molecular weight, presumably linked by a disulfide bridge(s)

  19. Acetylcholine induces GABA release onto rod bipolar cells through heteromeric nicotinic receptors expressed in A17 amacrine cells

    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 Ca2+ accumulation in A17 cell dendrites and varicosities mediated by L-type voltage-gated calcium channels (VGCCs) and intracellular Ca2+ 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. PMID:25709566

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

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

    2011-01-01

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

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

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

    2009-01-01

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

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

    Paes, Paulo Cesar de Arruda

    2002-07-01

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

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

    Jung Jin Lee

    2014-01-01

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

  4. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne;

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development,...

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

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

    1995-01-01

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

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

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

    2007-01-01

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

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

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

    2010-01-01

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

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

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

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

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

    1990-10-01

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

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

    Avi Ring

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

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

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

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

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

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

    2012-01-01

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

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

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

    2010-01-01

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

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

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

    1997-01-01

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

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

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

    2002-01-01

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

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

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

    2009-03-01

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

  17. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

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

    Jennifer L Hellier

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

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

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

    2016-06-01

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

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

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

    1980-01-01

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

  1. [3H]imidacloprid: synthesis of a candidate radioligand for the nicotinic acetylcholine receptor

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

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

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

    2016-05-01

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

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

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

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

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

    2013-01-01

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

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

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

    2015-09-01

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

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

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

    2010-01-01

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

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

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

  8. In vitro evaluation of nicotinic acetylcholine receptors with 2-[18F]F-A85380 in Parkinson's disease

    Nicotinic acetylcholine receptors (nAChR) are involved in many physiological functions and appear to be affected in neurodegenerative diseases like Alzheimer's disease and Parkinson's disease (PD). Here, we describe the in vitro evaluation of nAChRs in PD with 2-[18F]F-A85380, a ligand with high affinity to the β2 nAChR subunit. Autoradiography with 2-[18F]F-A85380 in untreated rat brain corresponded to the known distribution of α4β2 nAChRs with high uptake in the thalamus, moderate uptake in the striatum and cortex and low uptake in the cerebellum (47%, 43% and 19% of the thalamus, respectively). The localization of α4β2 nAChRs in the striatum was investigated in rodents with unilateral lesion of the substantia nigra. 2-[18F]F-A85380 binding was significantly reduced in the striatum ipsilateral to the lesion side (to 64% of the contralateral side), indicating that a fraction of α4β2 nAChRs is located on dopaminergic terminals, whereas another fraction resides on striatal interneurons or cortical afferents. Similarly, in human brain sections of PD patients, 2-[18F]F-A85380 uptake was significantly reduced not only in the caudate and putamen but also in the thalamus (approximately 30% of the binding of control brain in all three regions); within the striatum, nAChRs in the putamen were significantly more severely affected as in the caudate. The observed pattern of α4β2* nAChR loss demonstrates the potential of 2-[18F]F-A85380 for further investigations of this positron emission tomography ligand for in vivo studies of α4β2* nAChRs in PD

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

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

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

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

    1982-01-01

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

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

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

    2016-03-01

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

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

    Marlet Martinez-Archundia

    2012-01-01

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

  13. Mice lacking the alpha4 nicotinic receptor subunit fail to modulate dopaminergic neuronal arbors and possess impaired dopamine transporter function.

    Parish, C L; Nunan, J; Finkelstein, D I; McNamara, F N; Wong, J Y; Waddington, J L; Brown, R M; Lawrence, A J; Horne, M K; Drago, J

    2005-11-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) at presynaptic sites can modulate dopaminergic synaptic transmission by regulating dopamine (DA) release and uptake. Dopaminergic transmission in nigrostriatal and mesolimbic pathways is vital for the coordination of movement and is associated with learning and behavioral reinforcement. We reported recently that the D2 DA receptor plays a central role in regulating the arbor size of substantia nigra dopaminergic neurons. Given the known effects of nAChRs on dopaminergic neurotransmission, we assessed the ability of the alpha4 nAChR subunit to regulate arbor size of dopaminergic neurons by comparing responses of wild-type and alpha4 nAChR subunit knockout [alpha4(-/-)] mice to long-term exposure to cocaine, amphetamine, nicotine, and haloperidol, and after substantia nigra neurotoxic lesioning. We found that dopaminergic neurons in adult drug-naive alpha4(-/-) mice had significantly larger terminal arbors, and despite normal short-term behavioral responses to drugs acting on pre- and postsynaptic D2 DA receptors, they were unable to modulate their terminal arbor in response to pharmacological manipulation or after lesioning. In addition, although synaptosome DA uptake studies showed that the interaction of the D2 DA receptor and the dopamine transporter (DAT) was preserved in alpha4(-/-) mice, DAT function was found to be impaired. These findings suggest that the alpha4 subunit of the nAChR is an independent regulator of terminal arbor size of nigrostriatal dopaminergic neurons and that reduced functionality of presynaptic DAT may contribute to this effect by impairing DA uptake. PMID:16077034

  14. The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

    Wieskopf, Jeffrey S; Mathur, Jayanti; Limapichat, Walrati;

    2015-01-01

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified...... expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with...

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

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

    2011-01-01

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

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

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

    2013-12-01

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

  17. The 15q13.3 deletion syndrome: Deficient α(7)-containing nicotinic acetylcholine receptor-mediated neurotransmission in the pathogenesis of neurodevelopmental disorders.

    Deutsch, Stephen I; Burket, Jessica A; Benson, Andrew D; Urbano, Maria R

    2016-01-01

    Array comparative genomic hybridization (array CGH) has led to the identification of microdeletions of the proximal region of chromosome 15q between breakpoints (BP) 3 or BP4 and BP5 encompassing CHRNA7, the gene encoding the α7-nicotinic acetylcholine receptor (α7nAChR) subunit. Phenotypic manifestations of persons with these microdeletions are variable and some heterozygous carriers are seemingly unaffected, consistent with their variable expressivity and incomplete penetrance. Nonetheless, the 15q13.3 deletion syndrome is associated with several neuropsychiatric disorders, including idiopathic generalized epilepsy, intellectual disability, autism spectrum disorders (ASDs) and schizophrenia. Haploinsufficient expression of CHRNA7 in this syndrome has highlighted important roles the α7nAChR plays in the developing brain and normal processes of attention, cognition, memory and behavior throughout life. Importantly, the existence of the 15q13.3 deletion syndrome contributes to an emerging literature supporting clinical trials therapeutically targeting the α7nAChR in disorders such as ASDs and schizophrenia, including the larger population of patients with no evidence of haploinsufficient expression of CHRNA7. Translational clinical trials will be facilitated by the existence of positive allosteric modulators (PAMs) of the α7nAChR that act at sites on the receptor distinct from the orthosteric site that binds acetylcholine and choline, the receptor's endogenous ligands. PAMs lack intrinsic efficacy by themselves, but act where and when the endogenous ligands are released in response to relevant social and cognitive provocations to increase the likelihood they will result in α7nAChR ion channel activation. PMID:26257138

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

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

    2016-03-01

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

  19. Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

    To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

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

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

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

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

    2016-08-26

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

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

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

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

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

    2000-08-30

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

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

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

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

    Supavilai, P.; Karobath, M.

    1985-02-04

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

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

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

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

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

    2011-06-01

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

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

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

    2010-07-13

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

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

    Francesca Prestori

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

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

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

    2010-01-01

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

  11. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α2β2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α2β2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α2β2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α2β2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α2β2 insulin receptor subunit complex

  12. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

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

    2015-01-01

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

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

    Chiba, Tomohiro

    2010-01-01

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

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

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

    2012-01-01

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

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

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

    2008-01-01

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

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

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

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

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

    1981-11-26

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

  18. Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    glycine-free or a glycine-bound subunit. Agonist-free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (-) subunit interfaces. In heteromeric receptors comprising wild-type and R65K,T204A,R271C triple-mutant subunits, the fluorescence...... response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine-free subunits were activated by glycine binding to neighboring wild-type subunits, our results provide evidence for a cooperative...... activation mechanism. However, because the fluorescent label on glycine-free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non-concerted conformational change that is not essential for receptor activation....

  19. Role of desensitization and subunit expression for kainate receptor-mediated neurotoxicity in murine neocortical cultures

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

    1999-01-01

    The neurotoxic actions of kainate and domoate were studied in cultured murine neocortical neurons at various days in culture and found to be developmentally regulated involving three components of neurotoxicity: (1) toxicity via indirect activation of N-methyl-D-aspartate (NMDA) receptors, (2) to...... produced by kainate receptors in mature cultures. Examining the subunit expression of the kainate receptor subunits GluR6/7 and KA2 did, however, not reveal any major change during development of the cultures....

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

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

    2016-04-01

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

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

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

    2010-09-01

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

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

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

    2010-06-01

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

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

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

    2015-09-01

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

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

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

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

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

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

    Allison Anne Feduccia

    2012-08-01

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

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

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

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

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

    2014-01-01

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

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

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

    2009-06-01

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

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

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

    2009-01-01

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

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

    Harpsøe, Kasper; Ahring, Philip K; Christensen, Jeppe K; Jensen, Marianne L; Peters, Dan; Balle, Thomas

    2011-01-01

    the observation of two distinct agonist sensitivities. Using different expression ratios of mammalian a4 and ß2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (a4)(3)(ß2)(2) receptor. In addition to two high-sensitivity sites at...... a4ß2 interfaces, the (a4)(3)(ß2)(2) receptor contains a third low-sensitivity agonist binding site in the a4a4 interface. Occupation of this site is required for full activation and is responsible for the widened dynamic response range of this receptor subtype. By site-directed mutagenesis, we show...... that three residues, which differ between the a4ß2 and a4a4 sites, control agonist sensitivity. The results presented here provide a basic insight into the function of pentameric ligand-gated ion channels, which enables modulation of the receptors with hitherto unseen precision; it becomes possible to...

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

    Malin Jonsson Fagerlund

    2016-02-01

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

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

    Yi Miao

    2013-02-01

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

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

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

    2016-06-15

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

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

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

    1999-01-01

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

  16. The stoichiometry of P2X2/6 receptor heteromers depends on relative subunit expression levels

    Barrera, Nelson P.; Henderson, Robert M.; Murrell-Lagnado, Ruth D.; Edwardson, J Michael

    2007-01-01

    Fast synaptic transmission involves the operation of ionotropic receptors, which are often composed of at least two types of subunit. We have developed a method, based on atomic force microscopy imaging to determine the stoichiometry and subunit arrangement within ionotropic receptors. We showed recently that the P2X(2) receptor for ATP is expressed as a trimer but that the P2X(6) subunit is unable to oligomerize. In this study we addressed the subunit stoichiometry of heteromers containing b...

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

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

    2014-01-01

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

  18. The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy.

    Andrea eBecchetti

    2015-02-01

    Full Text Available Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE is a focal epilepsy with attacks typically arising in the frontal lobe during non rapid eye movement (NREM sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs. This is consistent with the widespread expression of these receptors, particularly the α4β2* subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na+-dependent K+ channel, DEPD5 (Dishevelled, Egl-10 and Pleckstrin Domain-containing protein 5, and CRH (Corticotropin-Releasing Hormone. Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.

  19. Positive modulation of delta-subunit containing GABAA receptors in mouse neurons

    Vardya, Irina; Hoestgaard-Jensen, Kirsten; Nieto-Gonzalez, Jose Luis;

    2012-01-01

    δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA(A) recep......δ-subunit containing extrasynaptic GABA(A) receptors are potential targets for modifying neuronal activity in a range of brain disorders. With the aim of gaining more insight in synaptic and extrasynaptic inhibition, we used a new positive modulator, AA29504, of δ-subunit containing GABA......-free environment using Ca²⁺ imaging in cultured neurons, AA29504 showed GABA(A) receptor agonism in the absence of agonist. Finally, AA29504 exerted dose-dependent stress-reducing and anxiolytic effects in mice in vivo. We propose that AA29504 potentiates δ-containing GABA(A) receptors to enhance tonic inhibition...

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

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

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

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

    1990-12-14

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

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

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

    2010-09-01

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

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

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

    2011-09-01

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

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

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

    2016-09-01

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

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

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

    2009-01-01

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

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

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

    2010-05-01

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

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

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

    2011-06-14

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

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

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

    2015-01-01

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

  9. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    Molinari, A M; Abbondanza, C; Armetta, I.; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-01-01

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl...

  10. Expression of nicotinic acetylcholine receptor subunit α9 in type Ⅱ vestibular hair cells of rats

    Wei-jia KONG; Hua-mao CHENG; Paul van CAUWENBERGE

    2006-01-01

    Aim: To explore the cell specific existence of α9 AChR in the vestibular type Ⅱ hair cells (VHC Ⅱ) of rats. Methods: To detect the expression of α9 AChR messenger RNA (mRNA) in the vestibular endorgans and single VHC Ⅱ of rats by using the reverse transcription polymerase chain reaction (RT-PCR) technique and the single cell RT-PCR technique, respectively. Results: It was shown that α9 AChR mRNA was detected in the vestibular endorgans. By using single-cell RT-PCR, mRNA encoding α9 AChR was also detected in the VHC Ⅱ of the rats. Sequence analysis of the PCR products confirmed identity to corresponding cDNA sequence in the predicted region. Conclusion: We established a method which could effectively detect the cell specific expression of mRNA in an individual VHC. Present data confirm that α9 AChR mRNA is expressed in the VHC Ⅱ of rats and indicates that α9 AChR may function as a mediator of efferent cholinergic signaling in mammalian VHC.

  11. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells.

    Watanabe, Kanako; Kanno, Takeshi; Oshima, Tadayuki; Miwa, Hiroto; Tashiro, Chikara; Nishizaki, Tomoyuki

    2008-03-01

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-d-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist dl-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G(1) phase of cell cycling and decreased the proportion in the S/G(2) phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G(1) phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling. PMID:18178157

  12. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

    The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression of the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G1 phase of cell cycling and decreased the proportion in the S/G2 phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G1 phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling

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

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

    2016-01-01

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

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

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

    2016-03-01

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

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

    Hai-peng Huang

    2016-01-01

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

  16. Artemin growth factor increases nicotinic cholinergic receptor subunit expression and activity in nociceptive sensory neurons

    Albers, Kathryn M.; Zhang, Xiu Lin; Diges, Charlotte M.; Schwartz, Erica S.; Yang, Charles I; Davis, Brian M.; Gold, Michael S.

    2014-01-01

    Background Artemin (Artn), a member of the glial cell line-derived growth factor (GDNF) family, supports the development and function of a subpopulation of peptidergic, TRPV1-positive sensory neurons. Artn (enovin, neublastin) is elevated in inflamed tissue and its injection in skin causes transient thermal hyperalgesia. A genome wide expression analysis of trigeminal ganglia of mice that overexpress Artn in the skin (ART-OE mice) showed elevation in nicotinic acetylcholine receptor (nAChR) s...

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

    Wenz, Jorge J; Barrantes, Francisco J

    2005-01-11

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

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

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

  19. Further characterization of the subunits of the receptor with high affinity for immunoglobulin E

    The α, β, γ subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the α chain as assessed from the incorporation studies. The results combined with future sequence data will permit unambiguous determination of the multiplicity of the chains in the receptor. Chymotryptic peptide maps of the extrinsically iodinated subunits show several similar peptides, particularly for α and β. However, these putative homologies were not apparent when tryptic maps of the biosynthetically ([3H]leucine) labeled subunits were analyzed

  20. Activated radixin is essential for GABAA receptor α5 subunit anchoring at the actin cytoskeleton

    Loebrich, Sven; Bähring, Robert; Katsuno, Tatsuya; Tsukita, Sachiko; Kneussel, Matthias

    2006-01-01

    Neurotransmitter receptor clustering is thought to represent a critical parameter for neuronal transmission. Little is known about the mechanisms that anchor and concentrate inhibitory neurotransmitter receptors in neurons. GABAA receptor (GABAAR) α5 subunits mainly locate at extrasynaptic sites and are thought to mediate tonic inhibition. Notably, similar as synaptic GABAARs, these receptor subtypes also appear in cluster formations at neuronal surface membranes and are of particular interes...

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

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

    2015-09-01

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

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

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

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

    Dezaki, Katsuya; Kimura, Ikuko

    1998-01-01

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

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

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

    2007-01-01

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

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

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

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

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

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

    2009-01-01

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

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

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

    2014-10-01

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

  8. Disassembly of the cholinergic postsynaptic apparatus induced by axotomy in mouse sympathetic neurons: the loss of dystrophin and beta-dystroglycan immunoreactivity precedes that of the acetylcholine receptor.

    Zaccaria, M L; De Stefano, M E; Properzi, F; Gotti, C; Petrucci, T C; Paggi, P

    1998-08-01

    In mouse sympathetic superior cervical ganglion (SCG), cortical cytoskeletal proteins such as dystrophin (Dys) and beta1sigma2 spectrin colocalize with beta-dystroglycan (beta-DG), a transmembrane dystrophin-associated protein, and the acetylcholine receptor (AChR) at the postsynaptic specialization. The function of the dystrophin-dystroglycan complex in the organization of the neuronal cholinergic postsynaptic apparatus was studied following changes in the immunoreactivity of these proteins during the disassembly and subsequent reassembly of the postsynaptic specializations induced by axotomy of the ganglionic neurons. After axotomy, a decrease in the number of intraganglionic synapses was observed (t1/2 8 h 45'), preceded by a rapid decline of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3 AChR subunit (alpha3AChR) (t1/2 3 h 45', 4 h 30' and 6 h, respectively). In contrast, the percentage of postsynaptic densities immunopositive for beta1sigma2 spectrin remained unaltered. When the axotomized neurons began to regenerate their axons, the number of intraganglionic synapses increased, as did that of postsynaptic specializations immunopositive for beta-DG, Dys, and alpha3AChR. The latter number increased more slowly than that of Dys and beta-DG. These observations suggest that in SCG neurons, the dystrophin-dystroglycan complex might play a role in the assembly-disassembly of the postsynaptic apparatus, and is probably involved in the stabilization of AChR clusters. PMID:9720492

  9. A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

    S. Gattenlöhner

    2010-01-01

    Full Text Available Rhabdomyosarcoma (RMS is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR γ-subunit, which defines the fetal AChR (fAChR isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA. While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

  10. α7 Nicotinic acetylcholine receptor-specific antibody induces inflammation and amyloid β42 accumulation in the mouse brain to impair memory.

    Olena Lykhmus

    Full Text Available Nicotinic acetylcholine receptors (nAChRs expressed in the brain are involved in regulating cognitive functions, as well as inflammatory reactions. Their density is decreased upon Alzheimer disease accompanied by accumulation of β-amyloid (Aβ42, memory deficit and neuroinflammation. Previously we found that α7 nAChR-specific antibody induced pro-inflammatory interleukin-6 production in U373 glioblastoma cells and that such antibodies were present in the blood of humans. We raised a hypothesis that α7 nAChR-specific antibody can cause neuroinflammation when penetrating the brain. To test this, C57Bl/6 mice were either immunized with extracellular domain of α7 nAChR subunit α7(1-208 or injected with bacterial lipopolysaccharide (LPS for 5 months. We studied their behavior and the presence of α3, α4, α7, β2 and β4 nAChR subunits, Aβ40 and Aβ42 and activated astrocytes in the brain by sandwich ELISA and confocal microscopy. It was found that either LPS injections or immunizations with α7(1-208 resulted in region-specific decrease of α7 and α4β2 and increase of α3β4 nAChRs, accumulation of Aβ42 and activated astrocytes in the brain of mice and worsening of their episodic memory. Intravenously transferred α7 nAChR-specific-antibodies penetrated the brain parenchyma of mice pre-injected with LPS. Our data demonstrate that (1 neuroinflammation is sufficient to provoke the decrease of α7 and α4β2 nAChRs, Aβ42 accumulation and memory impairment in mice and (2 α7(1-208 nAChR-specific antibodies can cause inflammation within the brain resulting in the symptoms typical for Alzheimer disease.

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

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

  12. Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

    Hamouda, Ayman K; Stewart, Deirdre S; Chiara, David C; Savechenkov, Pavel Y; Bruzik, Karol S; Cohen, Jonathan B

    2014-05-01

    At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1β3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators. PMID:24563544

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

    Ren, Guilin Robin

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

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

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

    2009-01-01

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

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

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

    2015-09-01

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

  16. Insulin binding changes the interface region between α subunits of the insulin receptor

    The homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) was used to probe the interface region between the two α subunits of the α2β2 human insulin receptor. The two α subunits formed a covalent dimer when affinity-purified receptor or membrane-bound receptor was reacted with DSS. The α2 species was detected on protein blots from SDS gels using an anti-α-subunit antibody or 125I-concanavalin A. Alternatively, iodinated receptor was reacted with DSS and the α2 species measured directly in an SDS gel. As shown by all three assay systems, more α2 was formed when insulin was bound to receptor than when insulin was absent. These data indicate that the conformational change which occurs in the α subunit response to insulin binding results in a change in the α-α interaction within the receptor complex. The results are consistent with a kinase activation mechanism involving communication between the two αβ receptor halves

  17. Subetta increases phosphorylation of insulin receptor β-subunit alone and in the presence of insulin

    Gorbunov, E A; Nicoll, J; Kachaeva, E. V.; Tarasov, S A; Epstein, O. I.

    2015-01-01

    It has been previously shown that Subetta (a drug containing released-active forms of antibodies to the insulin receptor β-subunit and antibodies to endothelial nitric oxide synthase) stimulated insulin-induced adiponectin production by mature human adipocytes in the absence of insulin. Therefore, it was assumed that Subetta could activate the insulin receptor. To confirm this hypothesis, the capacity of Subetta to activate the insulin receptor in mature human adipocytes in the absence or pre...

  18. Editing modifies the GABA(A) receptor subunit alpha3

    Ohlson, Johan; Pedersen, Jakob Skou; Haussler, David;

    2007-01-01

    to find selectively edited sites and combined it with bioinformatic techniques that find stem-loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the alpha3 subunit of the GABA...

  19. AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

    Nadine Harmel; Barbara Cokic; Gerd Zolles; Henrike Berkefeld; Veronika Mauric; Bernd Fakler; Valentin Stein; Nikolaj Klöcker

    2012-01-01

    Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining alpha-subunits GluA1-4 and auxiliary beta-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstit...

  20. DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke

    Tu, Weihong; Xu, Xin; Peng, Lisheng; Zhong, Xiaofen; Zhang, Wenfeng; Soundarapandian, Mangala M.; Balel, Cherine; Wang, Manqi; Jia, Nali; Zhang, Wen; Lew, Frank; Chan, Sic Lung; Chen, Yanfang; Lu, Youming

    2010-01-01

    SUMMARY N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage. PMID:20141836

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

    Rohde, Line Aagot Hede; Ahring, Philip Kiær; Jensen, Marianne Lerbech; Nielsen, Elsebet Østergaard; Peters, Dan; Helgstrand, Charlotte; Krintel, Christian; Harpsøe, Kasper; Gajhede, Michael; Kastrup, Jette Sandholm; Balle, Thomas

    2012-01-01

    efficacy of a compound appears tightly coupled to its ability to form a strong inter-subunit bridge linking the primary and complementary binding interfaces. For the tested agonists, a specific halogen bond was observed to play a large role in establishing such strong inter-subunit anchoring....

  2. Differential pharmacological activity of JN403 between α7 and muscle nicotinic acetylcholine receptors.

    Arias, Hugo R; De Rosa, Maria Jose; Bergé, Ignacio; Feuerbach, Dominik; Bouzat, Cecilia

    2013-11-26

    The differential action of the novel agonist JN403 at neuronal α7 and muscle nicotinic receptors (AChRs) was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations ~1000-fold lower and ~20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking the ε subunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α-δ but not at the α-ε binding site. JN403 does not elicit detectable Ca²⁺ influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [³H]cytisine and [³H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs. PMID:24164482

  3. Similar GABAA receptor subunit composition in somatic and axon initial segment synapses of hippocampal pyramidal cells.

    Kerti-Szigeti, Katalin; Nusser, Zoltan

    2016-01-01

    Hippocampal pyramidal cells (PCs) express many GABAAR subunit types and receive GABAergic inputs from distinct interneurons. Previous experiments revealed input-specific differences in α1 and α2 subunit densities in perisomatic synapses, suggesting distinct IPSC decay kinetics. However, IPSC decays evoked by axo-axonic, parvalbumin- or cholecystokinin-expressing basket cells were found to be similar. Using replica immunogold labeling, here we show that all CA1 PC somatic and AIS synapses contain the α1, α2, β1, β2, β3 and γ2 subunits. In CA3 PCs, 90% of the perisomatic synapses are immunopositive for the α1 subunit and all synapses are positive for the remaining five subunits. Somatic synapses form unimodal distributions based on their immunoreactivity for these subunits. The α2 subunit densities in somatic synapses facing Cav2.1 (i.e. parvalbumin) or Cav2.2 (cholecystokinin) positive presynaptic active zones are comparable. We conclude that perisomatic synapses made by three distinct interneuron types have similar GABAA receptor subunit content. PMID:27537197

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

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

    2004-01-01

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

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

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

    2015-10-01

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

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

    Denis Kudryavtsev

    2015-05-01

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

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

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

    2012-01-01

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

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

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

    2011-01-01

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

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

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

    2010-01-01

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

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

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

    2002-01-01

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

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

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

    2009-01-01

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

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

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

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

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

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

    2014-01-01

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

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

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

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

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

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

    1994-01-01

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

  16. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  17. Purification and subunit composition of atrial natriuretic peptide receptor.

    Schenk, D B; Phelps, M N; Porter, J G; Fuller, F; Cordell, B; Lewicki, J A

    1987-01-01

    A receptor for atrial natriuretic peptide (ANP) was purified 2700-fold, to apparent homogeneity, from cultured bovine aortic smooth muscle cells by affinity chromatography. The native ANP receptor has a molecular weight of 125,000 as determined by both metrizamide gradient centrifugation and nonreducing NaDodSO4/polyacrylamide gel electrophoresis. With 125I-labeled ANP as ligand, the purified receptor bound a maximum of 5.70 nmol of ligand per mg of protein and the dissociation constant was 4...

  18. A negative allosteric modulator modulates GABAB-receptor signalling through GB2 subunits.

    Sun, Bing; Chen, Linhai; Liu, Lei; Xia, Zhixiong; Pin, Jean-Philippe; Nan, Fajun; Liu, Jianfeng

    2016-03-15

    An γ-aminobutyric acid type B (GABAB)-receptor mediates slow and prolonged synaptic inhibition in the central nervous system, which represents an interesting target for the treatment of various diseases and disorders of the central nervous system. To date, only one activator of the GABAB-receptor, baclofen, is on the market for the treatment of spasticity. Inhibitors of the GABAB-receptor, such as antagonists, show anti-absence seizure activity and pro-cognitive properties. In a search for allosteric compounds of the GABAB-receptor, although several positive allosteric modulators have been developed, it is only recently that the first negative allosteric modulator (NAM), CLH304a (also named Compound 14), has been reported. In the present study, we provide further information on the mechanism of action of CLH304a, and also show the possibility of designing more NAMs, such as CLH391 and CLH393, based on the structure of CLH304a. First we show that CLH304a inhibits native GABAB-receptor activity in cultured cerebellar granular neurons. We then show that CLH304a has inverse agonist properties and non-competitively inhibits the effect of agonists, indicating that it binds at a different site to GABA. The GABAB-receptor is a mandatory heterodimer made of GB1 subunits, in which agonists bind, and GB2 subunits, which activate G-proteins. By using various combinations made up of wild-type and/or mutated GB1 and GB2 subunits, we show that CLH304a acts on the heptahelical domain of GB2 subunits. These data revealed the possibility of designing innovative NAMs acting in the heptahelical domain of the GB2 subunits, offering novel possibilities for therapeutic intervention based on GABAB-receptor inhibition. PMID:26772870

  19. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl2) 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 HgCl2 and MeHg on [3H]-quinuclidinyl benzilate ([3H]-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 HgCl2 or MeHg to derive IC50 values and inhibition constants (K i). Results demonstrate that HgCl2 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 [3H]-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

  20. It's not “either/or”: activation and desensitization of nicotinic acetylcholine receptors both contribute to behaviors related to nicotine addiction and mood

    Picciotto, Marina R.; Addy, Nii A.; Mineur, Yann S.; Brunzell, Darlene H.

    2007-01-01

    Nicotine can both activate and desensitize/inactivate nicotinic acetylcholine receptors (nAChRs). An ongoing controversy in the field is to what extent the behavioral effects of nicotine result from activation of nAChRs, and to what extent receptor desensitization is involved in these behavioral processes. Recent electrophysiological studies have shown that both nAChR activation and desensitization contribute to the effects of nicotine in the brain, and these experiments have provided cellula...

  1. Identifying Barbiturate Binding Sites in a Nicotinic Acetylcholine Receptor with [3H]Allyl m-Trifluoromethyldiazirine Mephobarbital, a Photoreactive Barbiturate

    Hamouda, Ayman K.; Stewart, Deirdre S.; Chiara, David C.; Savechenkov, Pavel Y.; Bruzik, Karol S.; Cohen, Jonathan B.

    2014-01-01

    At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [3H]R-mTFD-MPAB ([3H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding si...

  2. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  3. Human locus coeruleus neurons express the GABAA receptor γ2 subunit gene and produce benzodiazepine binding

    Hellsten, Kati S.; Sinkkonen, Saku T.; Hyde, Thomas M.; Kleinman, Joel E.; Särkioja, Terttu; Maksimow, Anu; Uusi-Oukari, Mikko; Korpi, Esa R.

    2010-01-01

    Noradrenergic neurons of the locus coeruleus project throughout the cerebral cortex and multiple subcortical structures. Alterations in the locus coeruleus firing are associated with vigilance states and with fear and anxiety disorders. Brain ionotropic type A receptors for γ-aminobutyric acid (GABA) serve as targets for anxiolytic and sedative drugs, and play an essential regulatory role in the locus coeruleus. GABAA receptors are composed of a variable array of subunits forming heteropentam...

  4. The role of α7 nicotinic acetylcholine receptor in modulation of heart rate dynamics in endotoxemic rats.

    Mazloom, Roham; Eftekhari, Golnar; Rahimi-Balaei, Maryam; Rahimi, Maryam; Khori, Vahid; Hajizadeh, Sohrab; Dehpour, Ahmad R; Mani, Ali R

    2013-01-01

    Previous reports have indicated that artificial stimulation of the vagus nerve reduces systemic inflammation in experimental models of sepsis. This phenomenon is a part of a broader cholinergic anti-inflammatory pathway which activates the vagus nerve to modulate inflammation through activation of alpha7 nicotinic acetylcholine receptors (α7nACHR). Heart rate variability represents the complex interplay between autonomic nervous system and cardiac pacemaker cells. Reduced heart rate variability and increased cardiac cycle regularity is a hallmark of clinical conditions that are associated with systemic inflammation (e.g. endotoxemia and sepsis). The present study was aimed to assess the role of α7nACHR in modulation of heart rate dynamics during systemic inflammation. Systemic inflammation was induced by injection of endotoxin (lipopolysaccharide) in rats. Electrocardiogram and body temperature were recorded in conscious animals using a telemetric system. Linear and non-linear indices of heart rate variability (e.g. sample entropy and fractal-like temporal structure) were assessed. RT-PCR and immunohistochemistry studies showed that α7nACHR is expressed in rat atrium and is mainly localized at the endothelial layer. Systemic administration of an α7nACHR antagonist (methyllycaconitine) did not show a significant effect on body temperature or heart rate dynamics in naïve rats. However, α7nACHR blockade could further reduce heart rate variability and elicit a febrile response in endotoxemic rats. Pre-treatment of endotoxemic animals with an α7nACHR agonist (PHA-543613) was unable to modulate heart rate dynamics in endotoxemic rats but could prevent the effect of endotoxin on body temperature within 24 h experiment. Neither methyllycaconitine nor PHA-543613 could affect cardiac beating variability of isolated perfused hearts taken from control or endotoxemic rats. Based on our observations we suggest a tonic role for nicotinic acetylcholine receptors in

  5. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory.

    Butcher, Adrian J; Bradley, Sophie J; Prihandoko, Rudi; Brooke, Simon M; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M; Bottrill, Andrew R; Challiss, R A John; Broad, Lisa M; Felder, Christian C; Tobin, Andrew B

    2016-04-22

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser(228)) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser(228) These data supported the hypothesis that phosphorylation at Ser(228) was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser(228) on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser(228) phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser(228) not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  6. An Antibody Biosensor Establishes the Activation of the M1 Muscarinic Acetylcholine Receptor during Learning and Memory*♦

    Butcher, Adrian J.; Bradley, Sophie J.; Prihandoko, Rudi; Brooke, Simon M.; Mogg, Adrian; Bourgognon, Julie-Myrtille; Macedo-Hatch, Timothy; Edwards, Jennifer M.; Bottrill, Andrew R.; Challiss, R. A. John; Broad, Lisa M.; Felder, Christian C.; Tobin, Andrew B.

    2016-01-01

    Establishing the in vivo activation status of G protein-coupled receptors would not only indicate physiological roles of G protein-coupled receptors but would also aid drug discovery by establishing drug/receptor engagement. Here, we develop a phospho-specific antibody-based biosensor to detect activation of the M1 muscarinic acetylcholine receptor (M1 mAChR) in vitro and in vivo. Mass spectrometry phosphoproteomics identified 14 sites of phosphorylation on the M1 mAChR. Phospho-specific antibodies to four of these sites established that serine at position 228 (Ser228) on the M1 mAChR showed extremely low levels of basal phosphorylation that were significantly up-regulated by orthosteric agonist stimulation. In addition, the M1 mAChR-positive allosteric modulator, 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, enhanced acetylcholine-mediated phosphorylation at Ser228. These data supported the hypothesis that phosphorylation at Ser228 was an indicator of M1 mAChR activation. This was further supported in vivo by the identification of phosphorylated Ser228 on the M1 mAChR in the hippocampus of mice following administration of the muscarinic ligands xanomeline and 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. Finally, Ser228 phosphorylation was seen to increase in the CA1 region of the hippocampus following memory acquisition, a response that correlated closely with up-regulation of CA1 neuronal activity. Thus, determining the phosphorylation status of the M1 mAChR at Ser228 not only provides a means of establishing receptor activation following drug treatment both in vitro and in vivo but also allows for the mapping of the activation status of the M1 mAChR in the hippocampus following memory acquisition thereby establishing a link between M1 mAChR activation and hippocampus-based memory and learning. PMID:26826123

  7. Hco-LGC-38 is novel nematode cys-loop GABA receptor subunit.

    Siddiqui, Salma Z; Brown, David D R; Accardi, Michael V; Forrester, Sean G

    2012-10-01

    We have identified and characterized a novel cys-loop GABA receptor subunit (Hco-LGC-38) from the parasitic nematode Haemonchus contortus. This subunit is present in parasitic and free-living nematodes and shares similarity to both the UNC-49 group of GABA receptor subunits from nematodes and the resistant to dieldrin (RDL) receptors of insects. Expression of the Hco-lgc-38 gene in Xenopus oocytes and subsequent electrophysiological analysis has revealed that the gene encodes a homomeric channel sensitive to GABA (EC(50) 19 μM) and the GABA analogue muscimol. The sensitivity of the Hco-LGC-38 channel to GABA is similar to reported values for the Drosophila RDL receptor whereas its lower sensitivity to muscimol is similar to nematode GABA receptors. Hco-LGC-38 is also highly sensitive to the channel blocker picrotoxin and moderately sensitive to fipronil and dieldrin. Homology modeling of Hco-LGC-38 and subsequent docking of GABA and muscimol into the binding site has uncovered several types of potential interactions with binding-site residues and overall appears to share similarity with models of other invertebrate GABA receptors. PMID:22940478

  8. Expression of NR1 subunit of NMDA receptor in schizophrenic hippocampus

    Vrajová, M.; Peková, S.; Klaschka, Jan; Majer, E.; Šťastný, F.; Bubeníková-Valešová, V.

    Elsevier. Roč. 23, Suppl. 2 (2008), S152. ISSN 0924-9338 R&D Projects: GA MZd(CZ) NR8792 Institutional research plan: CEZ:AV0Z10300504 Keywords : NMDA receptor * NR1 subunit * schizophrenia * post-mortem studies

  9. Changes in the expression of NR1 subunit of NMDA receptor in schizophrenic hippocampus

    Vrajová, M.; Peková, S.; Klaschka, Jan; Štastný, P.

    Elsevier. Roč. 17, Suppl. 1 (2007), s237-s238. ISSN 0924-977X. [ECNP Congress /20./. 13.10.2006-17.10.2006, Vienna] Institutional research plan: CEZ:AV0Z10300504 Keywords : schizophrenia * NMDA receptor * NR1 subunit * hippocampus * gender * antipsychotics

  10. Impaired Discrimination Learning in Mice Lacking the NMDA Receptor NR2A Subunit

    Brigman, Jonathan L.; Feyder, Michael; Saksida, Lisa M.; Bussey, Timothy J.; Mishina, Masayoshi; Holmes, Andrew

    2008-01-01

    N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice…

  11. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  12. Activation of α7 nicotinic acetylcholine receptors protects potentiated synapses from depotentiation during theta pattern stimulation in the hippocampal CA1 region of rats.

    Galvez, Bryan; Gross, Noah; Sumikawa, Katumi

    2016-06-01

    Long-term potentiation (LTP) shows memory-like consolidation and thus becomes increasingly resistant to disruption by low-frequency stimulation (LFS). However, it is known that nicotine application during LFS uniquely depotentiates consolidated LTP. Here, we investigated how nicotine contributes to the disruption of stabilized LTP in the hippocampal CA1 region. We found that nicotine-induced depotentiation is not due to masking LTP by inducing long-term depression and requires the activation of GluN2A-containing NMDARs. We further examined whether nicotine-induced depotentiation involves the reversal of LTP mechanisms. LTP causes phosphorylation of Ser-831 on GluA1 subunits of AMPARs that increases the single-channel conductance of AMPARs. This phosphorylation remained unchanged after depotentiation. LTP involves the insertion of new AMPARs into the synapse and the internalization of AMPARs is associated with dephosphorylation of Ser-845 on GluA1 and caspase-3 activity. Nicotine-induced depotentiation occurred without dephosphorylation of the Ser-845 and in the presence of a caspase-3 inhibitor. LTP is also accompanied by increased filamentous actin (F-actin), which controls spine size. Nicotine-induced depotentiation was prevented by jasplakinolide, which stabilizes F-actin, suggesting that nicotine depotentiates consolidated LTP by destabilizing F-actin. α7 nicotinic acetylcholine receptor (nAChR) antagonists mimicked the effect of nicotine and selective removal of hippocampal cholinergic input caused depotentiation in the absence of nicotine, suggesting that nicotine depotentiates consolidated LTP by inducing α7 nAChR desensitization. Our results demonstrate a new role for nicotinic cholinergic systems in protecting potentiated synapses from depotentiation by preventing GluN2A-NMDAR-mediated signaling for actin destabilization. PMID:26867505

  13. P2Y13 receptors mediate presynaptic inhibition of acetylcholine release induced by adenine nucleotides at the mouse neuromuscular junction.

    Guarracino, Juan F; Cinalli, Alejandro R; Fernández, Verónica; Roquel, Liliana I; Losavio, Adriana S

    2016-06-21

    It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y

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

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

    2011-01-01

    Since the first description of 5-HT3 receptors more than 50 years ago, there has been speculation about the molecular basis of their receptor heterogeneity. We have cloned the genes encoding novel 5-HT3 subunits 5-HT3C, 5-HT3D, and 5-HT3E and have shown that these subunits are able to form functional heteromeric receptors when coexpressed with the 5-HT3A subunit. However, whether these subunits are actually expressed in human tissue remained to be confirmed. In the current study, we performed...

  15. Novel acetylcholine and carbamoylcholine analogues

    Hansen, Camilla Petrycer; Jensen, Anders Asbjørn; Christensen, Jeppe K.;

    2008-01-01

    A series of carbamoylcholine and acetylcholine analogues were synthesized and characterized pharmacologically at neuronal nicotinic acetylcholine receptors (nAChRs). Several of the compounds displayed low nanomolar binding affinities to the alpha 4beta 2 nAChR and pronounced selectivity for this ...

  16. Analysis of NR3A receptor subunits in human native NMDA receptors

    Nilsson, Anna; Eriksson, Maria; Muly, E Chris;

    2007-01-01

    NR3A, representing the third class of NMDA receptor subunits, was first studied in rats, demonstrating ubiquitous expression in the developing central nervous system (CNS), but in the adult mainly expressed in spinal cord and some forebrain nuclei. Subsequent studies showed that rodent and non-human...... primate NR3A expression differs. We have studied the distribution of NR3A in the human CNS and show a widespread distribution of NR3A protein in adult human brain. NR3A mRNA and protein were found in all regions of the cerebral cortex, and also in the subcortical forebrain, midbrain and hindbrain. Only...... very low levels of NR3A mRNA and protein could be detected in homogenized adult human spinal cord, and in situ hybridization showed that expression was limited to ventral motoneurons. We found that NR3A is associated with NR1, NR2A and NR2B in adult human CNS, suggesting the existence of native NR1-NR2...

  17. Proteolytic activity of the purified hormone-binding subunit in the estrogen receptor.

    Molinari, A M; Abbondanza, C; Armetta, I; Medici, N; Minucci, S; Moncharmont, B; Nigro, V; Puca, G A

    1991-05-15

    The hormone-binding subunit of the calf uterus estradiol receptor was purified as a hormone-free molecule. Immunoaffinity chromatography with a specific monoclonal antibody was used as the final step. The purified subunit was specifically labeled by radioactive diisopropyl fluorophosphate. The diisopropyl fluorophosphate-labeled amino acid was serine. The purified receptor was able to release the fluorogenic or chromogenic group from synthetic peptides containing phenylalanine at the carboxyl terminus. This occurred only in the presence of estradiol and was hampered by aprotinin and diisopropyl fluorophosphate. Estradiol-dependent hydrolytic activity was also found in the eluate from gel slices after SDS/PAGE of purified receptor. This activity comigrated with the renaturable estradiol-binding activity. The estradiol antagonists 4-hydroxytamoxifen and ICI 164,384 as well as other steroid hormones were unable to activate this hydrolytic activity. PMID:1709742

  18. Differential Effects of Quercetin and Quercetin Glycosides on Human α7 Nicotinic Acetylcholine Receptor-Mediated Ion Currents.

    Lee, Byung-Hwan; Choi, Sun-Hye; Kim, Hyeon-Joong; Jung, Seok-Won; Hwang, Sung-Hee; Pyo, Mi-Kyung; Rhim, Hyewhon; Kim, Hyoung-Chun; Kim, Ho-Kyoung; Lee, Sang-Mok; Nah, Seung-Yeol

    2016-07-01

    Quercetin is a flavonoid usually found in fruits and vegetables. Aside from its antioxidative effects, quercetin, like other flavonoids, has a various neuropharmacological actions. Quercetin-3-O-rhamnoside (Rham1), quercetin-3-O-rutinoside (Rutin), and quercetin- 3-(2(G)-rhamnosylrutinoside (Rham2) are mono-, di-, and tri-glycosylated forms of quercetin, respectively. In a previous study, we showed that quercetin can enhance α7 nicotinic acetylcholine receptor (α7 nAChR)-mediated ion currents. However, the role of the carbohydrates attached to quercetin in the regulation of α7 nAChR channel activity has not been determined. In the present study, we investigated the effects of quercetin glycosides on the acetylcholine induced peak inward current (IACh) in Xenopus oocytes expressing the α7 nAChR. IACh was measured with a two-electrode voltage clamp technique. In oocytes injected with α7 nAChR copy RNA, quercetin enhanced IACh, whereas quercetin glycosides inhibited IACh. Quercetin glycosides mediated an inhibition of IACh, which increased when they were pre-applied and the inhibitory effects were concentration dependent. The order of IACh inhibition by quercetin glycosides was Rutin≥Rham1>Rham2. Quercetin glycosides-mediated IACh enhancement was not affected by ACh concentration and appeared voltage-independent. Furthermore, quercetin-mediated IACh inhibition can be attenuated when quercetin is co-applied with Rham1 and Rutin, indicating that quercetin glycosides could interfere with quercetin-mediated α7 nAChR regulation and that the number of carbohydrates in the quercetin glycoside plays a key role in the interruption of quercetin action. These results show that quercetin and quercetin glycosides regulate the α7 nAChR in a differential manner. PMID:27098860

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

    Brown, Derek [Department of Psychiatry, Stobhill Hospital, Glasgow (United Kingdom); Chisholm, Jennifer A.; Patterson, Jim; Wyper, David [Department of Clinical Physics, Southern General Hospital, Glasgow, G51 4TF (United Kingdom); Owens, Jonathan; Pimlott, Sally [Department of Clinical Physics, Western Infirmary, Glasgow (United Kingdom)

    2003-02-01

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

  20. Complex Control of GABA(A) Receptor Subunit mRNA Expression: Variation, Covariation, and Genetic Regulation

    Mulligan, Megan K.; Wang, Xusheng; Adler, Adrienne L.; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W.

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly...

  1. Association analysis of GABA receptor subunit genes on 5q33 with heroin dependence in a Chinese male population.

    Loh, E W; Tang, N L S; Lee, D T S; Liu, S I; Stadlin, Alfreda

    2007-06-01

    GABAA receptor subunit genes clustered on 5q33 play a role in the development of alcoholism and methamphetamine use disorder without psychosis. The present study explored the possible contribution of the same subunit genes to the development of heroin dependence. Single nucleotide polymorphisms (SNPs) of the GABAA receptor subunits GABRB2, GABRA6, GABRA1, and GABRG2 were examined in 178 male Han Chinese heroin-dependent and 170 male control subjects. A significant difference in allele frequency for the SNP rs211014 in the GABAAgamma2 receptor subunit gene between cases and controls was identified (P = 0.015). A possible mechanism for the involvement of the GABA receptor subunit genes on 5q33 in the development of heroin dependence is discussed. PMID:17440936

  2. Receptor-binding region in human choriogonadotropin/lutropin β subunit

    Synthetic fragments have not been widely used thus far to evaluate structure-activity relations in the glycoprotein hormones. The authors prepared a series of peptides representing the intercysteine loop sequence (residues 38-57) in human choriogonadotropin (hCG) and lutropin (hLH) β subunits, anticipating that it might be oriented toward the surface and accessible to receptors. The peptides were characterized chemically and tested for bioactivity by binding to rat ovarian membrane receptor and stimulation of Leydig cell testosterone production. The hCGβ-(38-57) and hLHβ-(38-57) peptides inhibited binding of 125I-labeled hCG half-maximally at 1.51 x 10-4 and 2.03 x 10-5 M, respectively, while other peptide hormones and fragments from elsewhere in the β subunit were inactive. Both peptides stimulated testosterone production, with half-maximal responses at 3.55 x 10-5 M (hCG) and 2.18 x 10-5 M (hLH). By radioimmunoassay with an antibody to thyroglobulin-conjugated hCGβ-(38-57) peptide, native hCG and β subunit were highly reactive, as were the reduced and carboxymethylated subunit and peptide. These results indicate that the 38-57 region of β subunit is exposed on the surface and constitutes a component in the receptor-binding domain for hCG and hLH. A region of amphipathic-helical structure in the 38-57 sequence may promote hormone-receptor interactions in a manner proposed for several other peptide hormones

  3. Different interaction between the agonist JN403 and the competitive antagonist methyllycaconitine with the human alpha7 nicotinic acetylcholine receptor.

    Arias, Hugo R; Gu, Ruo-Xu; Feuerbach, Dominik; Wei, Dong-Qing

    2010-05-18

    The interaction of the agonist JN403 with the human (h) alpha7 nicotinic acetylcholine receptor (AChR) was compared to that for the competitive antagonist methyllycaconitine (MLA). The receptor selectivity of JN403 was studied on the halpha7, halpha3beta4, and halpha4beta2 AChRs. The results established that the cationic center and the hydrophobic group found in JN430 and MLA are important for the interaction with the AChRs. MLA preincubation inhibits JN403-induced Ca(2+) influx in GH3-halpha7 cells with a potency 160-fold higher than that when MLA is co-injected with JN403. The most probable explanation, based on our dynamics results, is that MLA (more specifically the 3-methyl-2,5-dioxopyrrole ring and the B-D rings) stabilizes the resting conformational state. The order of receptor specificity for JN403 is as follows: halpha7 > halpha3beta4 ( approximately 40-fold) > halpha4beta2 ( approximately 500-fold). This specificity is based on a larger number of hydrogen bonds between the carbamate group (another pharmacophore) of JN403 and the halpha7 sites, the electrostatic repulsion between the positively charged residues around the halpha3beta4 sites and the cationic center of JN403, fewer hydrogen bonds for the interaction of JN403 with the halpha3beta4 AChR, and an unfavorable van der Waals interaction between JN403 and the alpha4-beta2 interface. The higher receptor specificity for JN403 could be important for the treatment of alpha7-related disorders, including dementias, pain-related ailments, depression, anxiety, and wound healing. PMID:20377277

  4. Radioligand imaging of α4β2* nicotinic acetylcholine receptors in Alzheimer’s disease and Parkinson’s disease

    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, [11C]nicotine PET and 5-[123I]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 (-)[18F]Flubatine or [18F]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.

  5. Subunit structure of the follitropin (FSH) receptor. Photoaffinity labeling of the membrane-bound receptor follitropin complex in situ

    Human follicle-stimulating hormone (hFSH) was acylated with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB) and radioiodinated (55 microCi/micrograms) for use as a photoaffinity probe to investigate the subunit structure of the FSH receptor in calf testis. After incubation with the photoaffinity probe and photolysis with UV light, the cross-linked hormone-receptor complex was solubilized from the membrane and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of the reducing agent dithiothreitol. Autoradiography of the polyacrylamide gels revealed two major bands, 64 kDa and 84 kDa. These were equivalent in molecular mass to those observed in a previous study in which performed hormone-receptor complexes were solubilized with detergent prior to formation of covalent cross-linkages through the use of homobifunctional cross-linking reagents. Reduction with dithiothreitol resulted in the loss of radioactivity from the 84-kDa band with a concomitant increase in the intensity of the 64-kDa band. Since dithiothreitol increases the dissociation of intact radioiodinated azidobenzoyl-FSH into subunits, it is suggested that the conversion of the 84-kDa band to the 64-kDa band by dithiothreitol is due to the loss of non-cross-linked hFSH subunit from the 84-kDa band and that the two bands observed after photoaffinity labeling arise from covalent bond formation between hFSH and a receptor subunit having a relative molecular weight (Mr) of 48,000. In addition to the predominant photolabeling of the receptor to yield the 64-kDa and 84-kDa bands, several other, less intense bands (54 kDa, 76 kDa, 97 kDa, and 116 kDa) were also consistently observed on autoradiographs

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

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

    2016-01-01

    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 [3H]-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...

  7. Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

    Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig;

    2010-01-01

    Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus......) conferred lesser nonspecific rate-suppressing effects, with no rate suppression for TBPB. In mutant mice lacking M(1) and M(4) receptors, xanomeline failed to diminish cocaine discrimination while rate-decreasing effects were intact. Our data suggest that central M(1) receptor activation attenuates cocaine...

  8. The Nicotinic α6 Subunit Gene Determines Variability in Chronic Pain Sensitivity via Cross-inhibition of P2X2/3 Receptors

    Wieskopf, Jeffrey S.; Mathur, Jayanti; Limapichat, Walrati; Post, Michael R.; Al-Qazzaz, Mona; Sorge, Robert E.; Martin, Loren J.; Zaykin, Dmitri V.; Smith, Shad B.; Freitas, Kelen; Austin, Jean-Sebastien; Dai, Feng; Zhang, Jie; Marcovitz, Jaclyn; Tuttle, Alexander H.; Slepian, Peter M.; Clarke, Sarah; Drenan, Ryan M.; Janes, Jeff; Sharari, Shakir Al; Segall, Samantha K.; Aasvang, Eske K.; Lai, Weike; Bittner, Reinhard; Richards, Christopher I.; Slade, Gary D.; Kehlet, Henrik; Walker, John; Maskos, Uwe; Changeux, Jean-Pierre; Devor, Marshall; Maixner, William; Diatchenko, Luda; Belfer, Inna; Dougherty, Dennis A.; Su, Andrew I.; Lummis, Sarah C.R.; Damaj, M. Imad; Lester, Henry A.; Patapoutian, Ardem; Mogil, Jeffrey S.

    2016-01-01

    Chronic pain is a highly prevalent and poorly managed human health problem. We used microarray-based expression genomics in 25 inbred mouse strains to identify dorsal root ganglion (DRG)-expressed genetic contributors to mechanical allodynia, a prominent symptom of chronic pain. We identified expression levels of Chrna6, which encodes the α6 subunit of the nicotinic acetylcholine receptor (nAChR), as highly associated with allodynia. We confirmed the importance of α6* (i.e., α6-containing) nAChRs by analyzing both gain- and loss-of-function mutants. We find that mechanical allodynia associated with neuropathic and inflammatory injuries is significantly altered in α6* mutants, and that α6* but not α4* nicotinic receptors are absolutely required for peripheral and/or spinal nicotine analgesia. Furthermore, we show that Chrna6’s role in analgesia is at least partially due to direct interaction and cross-inhibition of α6* nAChRs with P2X2/3 receptors in DRG nociceptors. Finally, we establish relevance of our results to humans by the observation of genetic association in patients suffering from chronic postsurgical pain and temporomandibular pain. PMID:25972004

  9. An assessment of radioimmunoassay procedures for determination of anti-acetylcholine receptor antibodies in the sera of patients with myasthenia gravis

    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)

  10. Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation

    Thomsen, Morgane; Conn, P Jeffrey; Lindsley, Craig;

    2010-01-01

    Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus....... More importantly, we tested the hypothesis that muscarinic receptor agonists with varied receptor subtype selectivity can blunt cocaine's discriminative stimulus and reinforcing effects; we hypothesized a critical role for the M(1) and/or M(4) receptor subtypes in this modulation. Mice were trained to...... discriminate cocaine from saline, or to self-administer intravenous cocaine chronically. The nonselective muscarinic antagonists scopolamine and methylscopolamine, the nonselective muscarinic agonists oxotremorine and pilocarpine, the M(1)/M(4)-preferring agonist xanomeline, the putative M(1)-selective agonist...

  11. Resolution of complex fluorescence spectra of lipids and nicotinic acetylcholine receptor by multivariate analysis reveals protein-mediated effects on the receptor's immediate lipid microenvironment

    Wenz, Jorge J; 10.1186/1757-5036-1-6

    2009-01-01

    Analysis of fluorescent spectra from complex biological systems containing various fluorescent probes with overlapping emission bands is a challenging task. Valuable information can be extracted from the full spectra, however, by using multivariate analysis (MA) of measurements at different wavelengths. We applied MA to spectral data of purified Torpedo nicotinic acetylcholine receptor (AChR) protein reconstituted into liposomes made up of dioleoylphosphatidic acid (DOPA) and dioleoylphosphatidylcholine (DOPC) doped with two extrinsic fluorescent probes (NBD-cholesterol/pyrene-PC). Forster resonance energy transfer (FRET) was observed between the protein and pyrene-PC and between pyrene-PC and NBD-cholesterol, leading to overlapping emission bands. Partial least squares analysis was applied to ...

  12. AMPA receptors commandeer an ancient cargo exporter for use as an auxiliary subunit for signaling.

    Nadine Harmel

    Full Text Available Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs. AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER. CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling.

  13. The Duration of Nicotine Withdrawal-Associated Deficits in Contextual Fear Conditioning Parallels Changes in Hippocampal High Affinity Nicotinic Acetylcholine Receptor Upregulation

    Gould, Thomas J.; Portugal, George S.; André, Jessica M.; Tadman, Matthew P.; Marks, Michael J.; Kenney, Justin W.; YILDIRIM, Emre; Adoff, Michael

    2012-01-01

    A predominant symptom of nicotine withdrawal is cognitive deficits, yet understanding of the neural basis for these deficits is limited. Withdrawal from chronic nicotine disrupts contextual learning in mice and this deficit is mediated by direct effects of nicotine in the hippocampus. Chronic nicotine treatment upregulates nicotinic acetylcholine receptors (nAChR); however, it is unknown whether upregulation is related to the observed withdawal-induced cognitive deficits. If a relationship be...

  14. Identification of Significant Association and Gene-Gene Interaction of GABA Receptor Subunit Genes in Autism

    Ma, D Q; Whitehead, P. L.; Menold, M M; Martin, E. R.; Ashley-Koch, A. E.; Mei, H; Ritchie, M. D.; Delong, G R; Abramson, R.K.; Wright, H. H.; Cuccaro, M. L.; Hussman, J. P.; Gilbert, J.R.; Pericak-Vance, M A

    2005-01-01

    Autism is a common neurodevelopmental disorder with a significant genetic component. Existing research suggests that multiple genes contribute to autism and that epigenetic effects or gene-gene interactions are likely contributors to autism risk. However, these effects have not yet been identified. Gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, has been implicated in autism etiology. Fourteen known autosomal GABA receptor subunit genes were studied...

  15. Antipsychotic-induced catalepsy is attenuated in mice lacking the M4 muscarinic acetylcholine receptor

    Fink-Jensen, Anders; Schmidt, Lene S; Dencker, Ditte;

    2011-01-01

    A delicate balance exists between the central dopaminergic and cholinergic neurotransmitter systems with respect to motor function. An imbalance can result in motor dysfunction as observed in Parkinson's disease patients and in patients treated with antipsychotic compounds. Cholinergic receptor a...

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

    Jensen, Anders A.; Gharagozloo, Parviz; Birdsall, Nigel J M;

    2006-01-01

    of tertiary and quaternary analogues as well as bisquaternary dimers of strychnine and brucine at human alpha1 and alpha1beta glycine receptors and at a chimera consisting of the amino-terminal domain of the alpha7 nicotinic receptor (containing the orthosteric ligand binding site) and the ion...... profiles of strychnine and brucine, none of the analogues displayed significant selectivity between the alpha1 and alpha1beta subtypes. The structure-activity relationships for the compounds at the alpha7/5-HT3 chimera were significantly different from those at the glycine receptors. Most strikingly......, quaternization of strychnine and brucine with substituents possessing different steric and electronic properties completely eliminated the activity at the glycine receptors, whereas binding affinity to the alpha7/5-HT3 chimera was retained for the majority of the quaternary analogues. This study provides an...

  17. Absence of prostaglandin E2-induced hyperalgesia in NMDA receptor ε subunit knockout mice

    Minami, Toshiaki; Sugatani, Junko; Sakimura, Kenji; Abe, Manabu; Mishina, Masayoshi; Ito, Seiji

    1997-01-01

    We have previously found that intrathecal administration of prostaglandins E2 (PGE2) and D2 (PGD2) into conscious mice induced hyperalgesia by the hot plate test. The present study investigated the involvement of N-methyl-D-aspartate (NMDA) receptor in the prostaglandin-induced hyperalgesia by use of mice lacking NMDA receptor ε1, ε4, or ε1/ε4 subunits.PGE2 induced hyperalgesia over a wide range of doses from 50 pg to 500 ng kg−1 in wild-type mice. But PGE2 could not induce hyperalgesia in ε1...

  18. Antipsychotic-induced catalepsy is attenuated in mice lacking the M4 muscarinic acetylcholine receptor

    Fink-Jensen, Anders; Schmidt, Lene S.; Dencker, Ditte; Schülein, Christina; Wess, Jürgen; Wörtwein, Gitta; Woldbye, David P.D.

    2011-01-01

    A delicate balance exists between the central dopaminergic and cholinergic neurotransmitter systems with respect to motor function. An imbalance can result in motor dysfunction as observed in Parkinson’s disease patients and in patients treated with antipsychotic compounds. Cholinergic receptor antagonists can alleviate extrapyramidal symptoms in Parkinson’s disease and motor side effects induced by antipsychotics. The effects of anticholinergics are mediated by muscarinic receptors of which ...

  19. NMDA receptor subunit expression and PAR2 receptor activation in colospinal afferent neurons (CANs during inflammation induced visceral hypersensitivity

    Caudle Robert M

    2009-09-01

    Full Text Available Abstract Background Visceral hypersensitivity is a clinical observation made when diagnosing patients with functional bowel disorders. The cause of visceral hypersensitivity is unknown but is thought to be attributed to inflammation. Previously we demonstrated that a unique set of enteric neurons, colospinal afferent neurons (CANs, co-localize with the NR1 and NR2D subunits of the NMDA receptor as well as with the PAR2 receptor. The aim of this study was to determine if NMDA and PAR2 receptors expressed on CANs contribute to visceral hypersensitivity following inflammation. Recently, work has suggested that dorsal root ganglion (DRG neurons expressing the transient receptor potential vanilloid-1 (TRPV1 receptor mediate inflammation induced visceral hypersensitivity. Therefore, in order to study CAN involvement in visceral hypersensitivity, DRG neurons expressing the TRPV1 receptor were lesioned with resiniferatoxin (RTX prior to inflammation and behavioural testing. Results CANs do not express the TRPV1 receptor; therefore, they survive following RTX injection. RTX treatment resulted in a significant decrease in TRPV1 expressing neurons in the colon and immunohistochemical analysis revealed no change in peptide or receptor expression in CANs following RTX lesioning as compared to control data. Behavioral studies determined that both inflamed non-RTX and RTX animals showed a decrease in balloon pressure threshold as compared to controls. Immunohistochemical analysis demonstrated that the NR1 cassettes, N1 and C1, of the NMDA receptor on CANs were up-regulated following inflammation. Furthermore, inflammation resulted in the activation of the PAR2 receptors expressed on CANs. Conclusion Our data show that inflammation causes an up-regulation of the NMDA receptor and the activation of the PAR2 receptor expressed on CANs. These changes are associated with a decrease in balloon pressure in response to colorectal distension in non-RTX and RTX lesioned

  20. Effects of visual deprivation during brain development on expression of AMPA receptor subunits in rat’s hippocampus

    Sayyed Alireza Talaei

    2015-06-01

    Conclusion: Dark rearing of rats during critical period of brain development changes the relative expression and also arrangement of both AMPA receptor subunits, GluR1 and GluR2 in the hippocampus, age dependently.

  1. At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor.

    Otvos, Reka A; Mladic, Marija; Arias-Alpizar, Gabriela; Niessen, Wilfried M A; Somsen, Govert W; Smit, August B; Kool, Jeroen

    2016-06-01

    The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca(2+))-flux assay and high-resolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives. PMID:26738519

  2. Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice

    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 (p3H](-) quinuclidinylbenzilate ([3H](-)QNB), a specific but nonsubtype selective mAChR antagonist, [3H]pirenzepine ([3H]PZ), a specific M1 mAChR antagonist and [3H]11-2-[[2-[(diethylamino) methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, ([3H]AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (Kd) and density (Bmax) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) [3H](-)QNB binding suggests that SS have fewer mAChR's than LS in many areas. These differences may relate to their differential ethanol sensitivity

  3. The selective alpha7 nicotinic acetylcholine receptor agonist A-582941 activates immediate early genes in limbic regions of the forebrain

    Thomsen, M S; Mikkelsen, J D; Timmermann, D B;

    2008-01-01

    Due to the cognitive-enhancing properties of alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonists, they have attracted interest for the treatment of cognitive disturbances in schizophrenia. Schizophrenia typically presents in late adolescence or early adulthood. It is therefore important...... juvenile and adult rat forebrain using two markers, activity-regulated cytoskeleton-associated protein (Arc) and c-Fos, to map neuronal activity. Acute administration of A-582941 (1, 3, 10 mg/kg) induced a dose-dependent increase in Arc mRNA expression in the medial prefrontal cortex (mPFC) and the ventral...... in the mPFC, VO/LO, and shell of the nucleus accumbens, in both juvenile and adult rats. The A-582941-induced c-Fos protein expression was significantly greater in the mPFC and VO/LO of juvenile compared with adult rats. These data indicate that A-582941-induced alpha7 nAChR stimulation activates...

  4. A Multi-Route Model of Nicotine-Cotinine Pharmacokinetics, Pharmacodynamics and Brain Nicotinic Acetylcholine Receptor Binding in Humans

    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. Increased cocaine self-administration in M4 muscarinic acetylcholine receptor knockout mice

    Schmidt, Lene Sørensen; Thomsen, Morgane; Weikop, Pia;

    2011-01-01

    Rationale The reinforcing effects of cocaine are mediated by the mesolimbic dopamine system. Behavioral and neurochemical studies have shown that the cholinergic muscarinic M4 receptor subtype plays an important role in regulation of dopaminergic neurotransmission. Objectives Here we investigated...... for the first time the involvement of M4 receptors in the reinforcing effects of cocaine using chronic intravenous cocaine self-administration in extensively backcrossed M4 receptor knockout (M4 -/-) mice. Methods We evaluated acquisition of cocaine self-administration in experimentally naïve mice....... Both cocaine self-administration and food-maintained operant behavior were evaluated under fixed ratio 1 (FR 1) and progressive ratio (PR) schedules of reinforcement. In addition, cocaine-induced dopamine release and cocaine-induced hyperactivity were evaluated. Results M4 -/- mice earned significantly...

  6. Early evolution of ionotropic GABA receptors and selective regimes acting on the mammalian-specific theta and epsilon subunits.

    Christopher J Martyniuk

    Full Text Available BACKGROUND: The amino acid neurotransmitter GABA is abundant in the central nervous system (CNS of both invertebrates and vertebrates. Receptors of this neurotransmitter play a key role in important processes such as learning and memory. Yet, little is known about the mode and tempo of evolution of the receptors of this neurotransmitter. Here, we investigate the phylogenetic relationships of GABA receptor subunits across the chordates and detail their mode of evolution among mammals. PRINCIPAL FINDINGS: Our analyses support two major monophyletic clades: one clade containing GABA(A receptor alpha, gamma, and epsilon subunits, and another one containing GABA(A receptor rho, beta, delta, theta, and pi subunits. The presence of GABA receptor subunits from each of the major clades in the Ciona intestinalis genome suggests that these ancestral duplication events occurred before the divergence of urochordates. However, while gene divergence proceeded at similar rates on most receptor subunits, we show that the mammalian-specific subunits theta and epsilon experienced an episode of positive selection and of relaxed constraints, respectively, after the duplication event. Sites putatively under positive selection are placed on a three-dimensional model obtained by homology-modeling. CONCLUSIONS: Our results suggest an early divergence of the GABA receptor subunits, before the split from urochordates. We show that functional changes occurred in the lineages leading to the mammalian-specific subunit theta, and we identify the amino acid sites putatively responsible for the functional divergence. We discuss potential consequences for the evolution of mammals and of their CNS.

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

    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...... (-)-dibenzoyl-D-tartaric acid as resolving agents. Enantiomer (R)-(-)-6¿a was found to be the eutomer, with K(i) values of 4.6 and 48.7 nM against rat and human a7 receptors, respectively....

  8. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    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.

  9. The non-competitive acetylcholinesterase inhibitor APS12-2 is a potent antagonist of skeletal muscle nicotinic acetylcholine receptors

    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 (IC50 = 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 (IC50 = 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 (α12β1γδ) muscle-type nicotinic acetylcholine receptors (nAChRs) have been incorporated (IC50 = 0.0005 μM), indicating a higher affinity of the compound for Torpedo (α12β1γδ) than for the mouse (α12β1γε) 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.

  10. 6-bromohypaphorine from marine nudibranch mollusk Hermissenda crassicornis is an agonist of human α7 nicotinic acetylcholine receptor.

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

    2015-03-01

    6-Bromohypaphorine (6-BHP) has been isolated from the marine sponges Pachymatisma johnstoni, Aplysina sp., and the tunicate Aplidium conicum, but data on its biological activity were not available. For the nudibranch mollusk Hermissenda crassicornis no endogenous compounds were known, and here we describe the isolation of 6-BHP from this mollusk and its effects on different nicotinic acetylcholine receptors (nAChR). Two-electrode voltage-clamp experiments on the chimeric α7 nAChR (built of 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. PMID:25775422

  11. 6-Bromohypaphorine from Marine Nudibranch Mollusk Hermissenda crassicornis is an Agonist of Human α7 Nicotinic Acetylcholine Receptor

    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.

  12. A novel inhibitor of α9α10 nicotinic acetylcholine receptors from Conus vexillum delineates a new conotoxin superfamily.

    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. Agonist actions of clothianidin on synaptic and extrasynaptic nicotinic acetylcholine receptors expressed on cockroach sixth abdominal ganglion.

    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. PMID:19583978

  14. GABAA receptor B subunit expression in the superior frontal cortex of human alcoholics

    Full text: Changes in GABAA receptor pharmacology can be ascribed to alterations in expression of specific GABAA receptor subunits. Ethanol is known to be a potent agonist of the GABAA receptor. Chronic abuse of alcohol in humans results in damage of selective brain regions such as the superior frontal cortex (SFC), leading to neuronal cell loss. Studies in our laboratory 1 and elsewhere 2 have shown differences in expression of a number of GABAA receptor subunits in chronic human alcoholism. This suggests that alterations in GABAA receptor composition may be involved in the pathogenesis of alcoholic brain damage. We analysed the expression of the β1 ,β2 and β3 isoforms of the GABAA receptor by a competitive reverse transcription polymerase chain reaction (RT-PCR) technique, which utilised an internal standard (IS) for quantitation. 35 S-dATP was incorporated to enable visualisation of the PCR products. Human brain tissue was obtained at autopsy and stored in 0.32 M sucrose at -80 deg C. Total RNA was extracted from pathologically susceptible and spared regions, SFC and motor cortex respectively,of 22 control and 22 alcoholic patients. 1 μg of total RNA from each sample was co-amplified with 0.5 pg of IS and a ratio determined. A standard consisting of known amounts of β1 cRNA titrated against 0.5 pg of IS enabled a standard curve to be generated for quantitation of each unknown sample. The samples were subjected to polyacrylamide gel electrophoresis and the dried gel exposed to a phosphorimager screen. Data analysis was performed using the ImageQuant program. Initial results indicate that there is a reduction in expression of all the β transcripts in alcoholics when compared with controls, which supports the hypothesis that the GABAA receptor is altered by alcohol abuse. Supported by NHMRC. Copyright (2001) Australian Neuroscience Society

  15. Annulated heterocyclic bioisosteres of norarecoline. Synthesis and molecular pharmacology at five recombinant human muscarinic acetylcholine receptors

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

    1995-01-01

    = 0.011 microM), and 4d (IC50 = 0.0008 microM). Pharmacological effects (EC50 or Ki values) and intrinsic activities (per cent of maximal carbachol responses) were determined using five recombinant human mAChRs (m1-m5) and the functional assay, receptor selection and amplification technology (R-SAT...

  16. A specific multi-nutrient formulation enhances M1 muscarinic acetylcholine receptor responses in vitro

    Savelkoul, P.J.M.; Janíčková, Helena; Kuipers, A.A.M.; Hageman, R.J.J.; Kamphuis, P.J.; Doležal, Vladimír; Broersen, L.M.

    2012-01-01

    Roč. 120, č. 4 (2012), s. 631-640. ISSN 0022-3042 R&D Projects: GA MŠk(CZ) 7E10060 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : muscarinic receptor * docosahexaenoic acid * Alzheimer’s disease Subject RIV: FH - Neurology Impact factor: 3.973, year: 2012

  17. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

    2002-01-01

    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  18. Discs-large (DLG is clustered by presynaptic innervation and regulates postsynaptic glutamate receptor subunit composition in Drosophila

    Featherstone David E

    2005-01-01

    Full Text Available Abstract Background Drosophila discs-large (DLG is the sole representative of a large class of mammalian MAGUKs, including human DLG, SAP 97, SAP102, and PSD-95. MAGUKs are thought to be critical for postsynaptic assembly at glutamatergic synapses. However, glutamate receptor cluster formation has never been examined in Drosophila DLG mutants. The fly neuromuscular junction (NMJ is a genetically-malleable model glutamatergic synapse widely used to address questions regarding the molecular mechanisms of synapse formation and growth. Here, we use immunohistochemistry, confocal microscopy, and electrophysiology to examine whether fly NMJ glutamate receptor clusters form normally in DLG mutants. We also address the question of how DLG itself is localized to the synapse by testing whether presynaptic innervation is required for postsynaptic DLG clustering, and whether DLG localization requires the presence of postsynaptic glutamate receptors. Results There are thought to be two classes of glutamate receptors in the Drosophila NMJ: 1 receptors that contain the subunit GluRIIA, and 2 receptors that contain the subunit GluRIIB. In DLG mutants, antibody staining for the glutamate receptor subunit GluRIIA is normal, but antibody staining for the glutamate receptor subunit GluRIIB is significantly reduced. Electrophysiological analysis shows an overall loss of functional postsynaptic glutamate receptors, along with changes in receptor biophysical properties that are consistent with a selective loss of GluRIIB from the synapse. In uninnervated postsynaptic muscles, neither glutamate receptors nor DLG cluster at synapses. DLG clusters normally in the complete absence of glutamate receptors. Conclusions Our results suggest that DLG controls glutamate receptor subunit composition by selectively stabilizing GluRIIB-containing receptors at the synapse. We also show that DLG, like glutamate receptors, is localized only after the presynaptic neuron contacts the

  19. Subtype-selective nicotinic acetylcholine receptor agonists can improve cognitive flexibility in an attentional set shifting task.

    Wood, Christopher; Kohli, Shivali; Malcolm, Emma; Allison, Claire; Shoaib, Mohammed

    2016-06-01

    Nicotinic acetylcholine receptors (nAChRs) are considered to be viable targets to enhance cognition in patients diagnosed with schizophrenia. Activation of nAChRs with selective nicotinic receptor agonists may provide effective means to pharmacologically treat cognitive deficits observed in schizophrenia. Cognitive flexibility is one aspect of cognition, which can be assessed in a rodent model of the attentional set-shifting task (ASST). The aim of the present study was two-fold, firstly, to evaluate the efficacy of a series of subtype selective nAChR agonists, such as those that target α7 and α4β2 nAChR subtypes in non-compromised rodents. Secondly, nicotine as a prototypic agonist was evaluated for its effects to restore attentional deficits produced by sub-chronic ketamine exposure in the ASST. Male hooded Lister rats underwent habituation, consisting of a simple odour and medium discrimination with subsequent assessment 24 h later. In experimentally naïve rats, α7 subtype selective agonists, compound-A and SSR180711 along with PNU-120596, an α7 positive allosteric modulator (PAM), were compared against the β2* selective agonist, 5IA-85380. All compounds except for PNU-120596 were observed to significantly improve extra-dimensional (ED) shift performance, nicotine, 5IA-85380 and SSR180711 further enhanced the final reversal (REV3) stage of the task. In another experiment, sub-chronic ketamine treatment produced robust deficits during the ED and the REV3 stages of the discriminations; rodents required significantly more trials to reach criterion during these discriminations. These deficits were attenuated in rodents treated acutely with nicotine (0.1 mg/kg SC) 10 min prior to the ED shift. These results highlight the potential utility of targeting nAChRs to enhance cognitive flexibility, particularly the α7 and β2* receptor subtypes. The improvement with nicotine was much greater in rodents that were impaired following the sub-chronic ketamine

  20. Studies for transitional changes of the muscarinic acetylcholine receptor and mRNA distribution by focal ischemia using nuclear medicine

    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 99mTc-hexametyl-propyleneamine oxime (99mTc-HMPAO), in vitro mAchR binding images with [3H] quinuclidinyl benzilate (3H-QNB), and mAchR-mRNA images by in situ hybridization method using 35S-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. The distribution of cerebral muscarinic acetylcholine receptors in vivo in patients with dementia. A controlled study with 123IQNB and single photon emission computed tomography

    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

  2. Nicotine Elevated Intracellular Ca2+ in Rat Airway Smooth Muscle Cells via Activating and Up-Regulating α7-Nicotinic Acetylcholine Receptor

    Yongliang Jiang

    2014-02-01

    Full Text Available Background: Chronic obstructive pulmonary disease (COPD is characterized by airway remodeling with airway smooth muscle (ASM hypertrophy and hyperplasia. Since tobacco use is the key risk factor for the development of COPD and intracellular Ca2+ concentration ([Ca2+]i plays a major role in both cell proliferation and differentiation, we hypothesized that nicotinic acetylcholine receptor (nAChR activation plays a role in the elevation of [Ca2+]i in airway smooth muscle cells (ASMCs. Methods: We examined the expression of nAChR and characterized the functions of α7-nAChR in ASMCs. Results: RT-PCR analysis showed that α2-7, β2, and β3-nAChR subunits are expressed in rat ASMCs, with α7 being one of the most abundantly expressed subtypes. Chronic nicotine exposure increased α7-nAChR mRNA and protein expression, and elevated resting [Ca2+]i in cultured rat ASMCs. Acute application of nicotine evoked a rapid increase in [Ca2+]i in a concentration-dependent manner, and the response was significantly enhanced in ASMCs cultured with 1 µM nicotine for 48 hours. Nicotine-induced Ca2+ response was reversibly blocked by the α7-nAChR nicotinic antagonists, methyllycaconitine and α-bungarotoxin. Small interfering RNA suppression of α7-nAChR also substantially blunted the Ca2+ responses induced by nicotine. Conclusion: These observations suggest that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.that nicotine elevates [Ca2+]i in ASMCs through α7-nAChR-mediated signals pathways, and highlight the possibility that α7-nAChR can be considered as a potential target for the treatment of airway remodeling.

  3. Evidence for cooperativity between nicotinic acetylcholine receptors in patch clamp records.

    Keleshian, A M; Edeson, R. O.; G.J. Liu; Madsen, B W

    2000-01-01

    It is often assumed that ion channels in cell membrane patches gate independently. However, in the present study nicotinic receptor patch clamp data obtained in cell-attached mode from embryonic chick myotubes suggest that the distribution of steady-state probabilities for conductance multiples arising from concurrent channel openings may not be binomial. In patches where up to four active channels were observed, the probabilities of two or more concurrent openings were greater than expected,...

  4. Molecular Mechanisms of Methoctramine Binding and Selectivity at Muscarinic Acetylcholine Receptors

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

    2014-01-01

    Roč. 86, č. 2 (2014), s. 180-192. ISSN 0026-895X R&D Projects: GA ČR(CZ) GAP304/12/0259; GA ČR(CZ) GA305/09/0681 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 ; RVO:61389030 Keywords : muscarinic receptors * selectivity * methoctramine * allosteric * FRET Subject RIV: ED - Physiology Impact factor: 4.128, year: 2014

  5. BLOCKADE OF CENTRAL NICOTINE ACETYLCHOLINE RECEPTOR SIGNALING ATTENUATE GHRELIN-INDUCED FOOD INTAKE IN RODENTS

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

    2010-01-01

    Here we sought to determine whether ghrelin's central effects on food intake can be interrupted by nicotinic cholinergic receptor (nAChR) blockade. Ghrelin regulates mesolimbic dopamine neurons projecting from the ventral tegmental area (VTA) to the nucleus accumbens (NAcc), 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 sou...

  6. Allosteric transitions of Torpedo acetylcholine receptor in lipids, detergent and amphipols

    Martinez, Karen L.; Gohon, Yann; Corringer, Pierre Jean;

    2002-01-01

    The binding of a fluorescent agonist to the acetycholine receptor from Torpedo electric organ has been studied by time-resolved spectroscopy in three different environments: in native membrane fragments, in the detergent CHAPS, and after complexation by amphipathic polymers ('amphipols'). Binding...... results from molecular interactions rather than from the loss of bulk physical properties of the membrane environment. © 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved....

  7. Inhibition of Toll-like receptor 2-mediated interleukin-8 production in Cystic Fibrosis airway epithelial cells via the alpha7-nicotinic acetylcholine receptor.

    Greene, Catherine M

    2010-01-01

    Cystic Fibrosis (CF) is an inherited disorder characterised by chronic inflammation of the airways. The lung manifestations of CF include colonization with Pseudomonas aeruginosa and Staphylococcus aureus leading to neutrophil-dominated airway inflammation and tissue damage. Inflammation in the CF lung is initiated by microbial components which activate the innate immune response via Toll-like receptors (TLRs), increasing airway epithelial cell production of proinflammatory mediators such as the neutrophil chemokine interleukin-8 (IL-8). Thus modulation of TLR function represents a therapeutic approach for CF. Nicotine is a naturally occurring plant alkaloid. Although it is negatively associated with cigarette smoking and cardiovascular damage, nicotine also has anti-inflammatory properties. Here we investigate the inhibitory capacity of nicotine against TLR2- and TLR4-induced IL-8 production by CFTE29o- airway epithelial cells, determine the role of alpha7-nAChR (nicotinic acetylcholine receptor) in these events, and provide data to support the potential use of safe nicotine analogues as anti-inflammatories for CF.

  8. Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons.

    Weber, Martin; Motin, Leonid; Gaul, Simon; Beker, Friederike; Fink, Rainer H A; Adams, David J

    2005-01-01

    The effects of intravenous (i.v.) anaesthetics on nicotinic acetylcholine receptor (nAChR)-induced transients in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and membrane currents were investigated in neonatal rat intracardiac neurons. In fura-2-loaded neurons, nAChR activation evoked a transient increase in [Ca(2+)](I), which was inhibited reversibly and selectively by clinically relevant concentrations of thiopental. The half-maximal concentration for thiopental inhibition of nAChR-induced [Ca(2+)](i) transients was 28 microM, close to the estimated clinical EC(50) (clinically relevant (half-maximal) effective concentration) of thiopental. In fura-2-loaded neurons, voltage clamped at -60 mV to eliminate any contribution of voltage-gated Ca(2+) channels, thiopental (25 microM) simultaneously inhibited nAChR-induced increases in [Ca(2+)](i) and peak current amplitudes. Thiopental inhibited nAChR-induced peak current amplitudes in dialysed whole-cell recordings by approximately 40% at -120, -80 and -40 mV holding potential, indicating that the inhibition is voltage independent. The barbiturate, pentobarbital and the dissociative anaesthetic, ketamine, used at clinical EC(50) were also shown to inhibit nAChR-induced increases in [Ca(2+)](i) by approximately 40%. Thiopental (25 muM) did not inhibit caffeine-, muscarine- or ATP-evoked increases in [Ca(2+)](i), indicating that inhibition of Ca(2+) release from internal stores via either ryanodine receptor or inositol-1,4,5-trisphosphate receptor channels is unlikely. Depolarization-activated Ca(2+) channel currents were unaffected in the presence of thiopental (25 microM), pentobarbital (50 microM) and ketamine (10 microM). In conclusion, i.v. anaesthetics inhibit nAChR-induced currents and [Ca(2+)](i) transients in intracardiac neurons by binding to nAChRs and thereby may contribute to changes in heart rate and cardiac output under clinical conditions. PMID:15644873

  9. Generation of functional inhibitory synapses incorporating defined combinations of GABA(A or glycine receptor subunits

    Christine Laura Dixon

    2015-12-01

    Full Text Available Fast inhibitory neurotransmission in the brain is mediated by wide range of GABAA receptor (GABAAR and glycine receptor (GlyR isoforms, each with different physiological and pharmacological properties. Because multiple isoforms are expressed simultaneously in most neurons, it is difficult to define the properties of inhibitory postsynaptic currents mediated by individual isoforms in vivo. Although recombinant expression systems permit the expression of individual isoforms in isolation, they require exogenous agonist application which cannot mimic the dynamic neurotransmitter profile characteristic of native synapses. We describe a neuron-HEK293 cell co-culture technique for generating inhibitory synapses incorporating defined combinations of GABAAR or GlyR subunits. Primary neuronal cultures, prepared from embryonic rat cerebral cortex or spinal cord, are used to provide presynaptic GABAergic and glycinergic terminals, respectively. When the cultures are mature, HEK293 cells expressing the subunits of interest plus neuroligin 2A are plated onto the neurons, which rapidly form synapses onto HEK293 cells. Patch clamp electrophysiology is then used to analyze the physiological and pharmacological properties of the inhibitory postsynaptic currents mediated by the recombinant receptors. The method is suitable for investigating the kinetic properties or the effects of drugs on inhibitory postsynaptic currents mediated by defined GABAAR or GlyR isoforms of interest, the effects of hereditary disease mutations on the formation and function of both types of synapses, and synaptogenesis and synaptic clustering mechanisms. The entire cell preparation procedure takes 2 – 5 weeks.

  10. Taurolithocholic acid promotes intrahepatic cholangiocarcinoma cell growth via muscarinic acetylcholine receptor and EGFR/ERK1/2 signaling pathway

    AMONYINGCHAROEN, SUMET; SURIYO, TAWIT; THIANTANAWAT, APINYA; WATCHARASIT, PIYAJIT; SATAYAVIVAD, JUTAMAAD

    2015-01-01

    Cholangiocarcinoma (CCA) is a malignant cancer of the biliary tract and its occurrence is associated with chronic cholestasis which causes an elevation of bile acids in the liver and bile duct. The present study aimed to investigate the role and mechanistic effect of bile acids on the CCA cell growth. Intrahepatic CCA cell lines, RMCCA-1 and HuCCA-1, were treated with bile acids and their metabolites to determine the growth promoting effect. Cell viability, cell cycle analysis, EdU incorporation assays were conducted. Intracellular signaling proteins were detected by western immunoblotting. Among eleven forms of bile acids and their metabolites, only taurolithocholic acid (TLCA) concentration dependently (1–40 μM) increased the cell viability of RMCCA-1, but not HuCCA-1 cells. The cell cycle analysis showed induction of cells in the S phase and the EdU incorporation assay revealed induction of DNA synthesis in the TLCA-treated RMCCA-1 cells. Moreover, TLCA increased the phosphorylation of EGFR, ERK 1/2 and also increased the expression of cyclin D1 in RMCCA-1 cells. Furthermore, TLCA-induced RMCCA-1 cell growth could be inhibited by atropine, a non-selective muscarinic acetylcholine receptor (mAChR) antagonist, AG 1478, a specific EGFR inhibitor, or U 0126, a specific MEK 1/2 inhibitor. These results suggest that TLCA induces CCA cell growth via mAChR and EGFR/EKR1/2 signaling pathway. Moreover, the functional presence of cholinergic system plays a certain role in TLCA-induced CCA cell growth. PMID:25815516

  11. Coronaridine congeners inhibit human α3β4 nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

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

    2015-08-01

    To characterize the interaction of coronaridine congeners with human (h) α3β4 nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The Ca(2+) influx results established that coronaridine congeners noncompetitively inhibit hα3β4 AChRs with the following potency (IC50's in μM) sequence: (-)-ibogamine (0.62±0.23)∼(+)-catharanthine (0.68±0.10)>(-)-ibogaine (0.95±0.10)>(±)-18-methoxycoronaridine [(±)-18-MC] (1.47±0.21)>(-)-voacangine (2.28±0.33)>(±)-18-methylaminocoronaridine (2.62±0.57 μM)∼(±)-18-hydroxycoronaridine (2.81±0.54)>(-)-noribogaine (6.82±0.78). A good linear correlation (r(2)=0.771) between the calculated IC50 values and their polar surface area was found, suggesting that this is an important structural feature for its activity. The radioligand competition results indicate that (±)-18-MC and (-)-ibogaine partially inhibit [(3)H]imipramine binding by an allosteric mechanism. Molecular docking, molecular dynamics, and in silico mutation results suggest that protonated (-)-18-MC binds to luminal [i.e., β4-Phe255 (phenylalanine/valine ring; position 13'), and α3-Leu250 and β4-Leu251 (leucine ring; position 9')], non-luminal, and intersubunit sites. The pharmacophore model suggests that nitrogens from the ibogamine core as well as methylamino, hydroxyl, and methoxyl moieties at position 18 form hydrogen bonds. Collectively our data indicate that coronaridine congeners inhibit hα3β4 AChRs by blocking the ion channel's lumen and probably by additional negative allosteric mechanisms by interacting with a series of non-luminal sites. PMID:26022277

  12. Angiotensin II type 2 receptor- and acetylcholine-mediated relaxation: essential contribution of female sex hormones and chromosomes.

    Pessôa, Bruno Sevá; Slump, Denise E; Ibrahimi, Khatera; Grefhorst, Aldo; van Veghel, Richard; Garrelds, Ingrid M; Roks, Anton J M; Kushner, Steven A; Danser, A H Jan; van Esch, Joep H M

    2015-08-01

    Angiotensin-induced vasodilation, involving type 2 receptor (AT2R)-induced generation of nitric oxide (NO; by endothelial NO synthase) and endothelium-derived hyperpolarizing factors, may be limited to women. To distinguish the contribution of female sex hormones and chromosomes to AT2R function and endothelium-derived hyperpolarizing factor-mediated vasodilation, we made use of the four-core genotype model, where the testis-determining Sry gene has been deleted (Y(-)) from the Y chromosome, allowing XY(-) mice to develop a female gonadal phenotype. Simultaneously, by incorporating the Sry gene onto an autosome, XY(-)Sry and XXSry transgenic mice develop into gonadal male mice. Four-core genotype mice underwent a sham or gonadectomy (GDX) operation, and after 8 weeks, iliac arteries were collected to assess vascular function. XY(-)Sry male mice responded more strongly to angiotensin than XX female mice, and the AT2R antagonist PD123319 revealed that this was because of a dilator AT2R-mediated effect occurring exclusively in XX female mice. The latter could not be demonstrated in XXSry male and XY(-) female mice nor in XX female mice after GDX, suggesting that it depends on both sex hormones and chromosomes. Indeed, treating C57bl/6 GDX male mice with estrogen could not restore angiotensin-mediated, AT2R-dependent relaxation. To block acetylcholine-induced relaxation of iliac arteries obtained from four-core genotype XX mice, both endothelial NO synthase and endothelium-derived hyperpolarizing factor inhibition were required, whereas in four-core genotype XY animals, endothelial NO synthase inhibition alone was sufficient. These findings were independent of gonadal sex and unaltered after GDX. In conclusion, AT2R-induced relaxation requires both estrogen and the XX chromosome sex complement, whereas only the latter is required for endothelium-derived hyperpolarizing factors. PMID:26056343

  13. Differences in muscarinic acetylcholine receptor subtypes in the central nervous system of long sleep and short sleep mice. [Ethanol effects

    Watson, M.; Ming, X.; McArdle, J.J. (Univ of Medical, Newark, NJ (USA))

    1989-02-09

    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<.01) greater. Binding assays for ({sup 3}H)(-) quinuclidinylbenzilate (({sup 3}H)(-)QNB), a specific but nonsubtype selective mAChR antagonist, ({sup 3}H)pirenzepine (({sup 3}H)PZ), a specific M1 mAChR antagonist and ({sup 3}H)11-2-((2-((diethylamino) methyl)-1-piperidinyl) acetyl)-5,11-dihydro-6H-pyrido (2,3-b) (1,4) benzodiazepine-6-one, (({sup 3}H)AF-DX 116), an M2 selective antagonist were performed to determine mAChR affinity (K{sub d}) and density (B{sub max}) in CNS regions such as the cerebral cortex, hippocampus, corpus striatum and other areas. Significantly lower (30-40%) ({sup 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.

  14. Different interaction between tricyclic antidepressants and mecamylamine with the human alpha3beta4 nicotinic acetylcholine receptor ion channel.

    Arias, Hugo R; Targowska-Duda, Katarzyna M; Feuerbach, Dominik; Sullivan, Carl J; Maciejewski, Ryszard; Jozwiak, Krzysztof

    2010-03-01

    The interaction of tricyclic antidepressants (TCAs) with the human (h)alpha3beta4 nicotinic acetylcholine receptor (AChR) in different conformational states was compared with that for mecamylamine by using functional and structural approaches including, Ca(2+) influx, radioligand binding, and molecular docking. The results established that: (a) [(3)H]imipramine binds to a single site with relatively high affinity (K(d) = 0.41 +/- 0.04 microM), (b) imipramine inhibits [(3)H]imipramine binding to the resting/kappa-bungarotoxin-bound AChR (K(i) = 0.68 +/- 0.08 microM) with practically the same affinity as to the desensitized/epibatidine-bound AChR (K(i) = 0.83 +/- 0.08 microM), suggesting that TCAs do not discriminate between these conformational states, and (c) although TCAs (IC(50) approximately 1.8-2.7 microM) and mecamylamine (IC(50) = 3.3 +/- 0.4 microM) inhibit (+/-)-epibatidine-induced Ca(2+) influx with potencies in the same concentration range, TCAs (K(i) approximately 1-3.6 microM), but not mecamylamine (apparent IC(50) approximately 0.2 mM), inhibit [(3)H]imipramine binding to halpha3beta4 AChRs in different conformational states. This is explained by our docking results where imipramine, in the neutral and protonated states, interacts with the leucine (position 9') and valine/phenylalanine (position 13') rings, whereas protonated mecamylamine (>99% at physiological pH) interacts with the outer ring (position 20'). Our data indicate that TCAs bind to overlapping sites located between the serine and valine/phenylalanine rings in the halpha3beta4 AChR ion channel, whereas protonated mecamylamine can be attracted to the channel mouth before blocking ion flux by interacting with a luminal site in its neutral state. PMID:20117161

  15. Assessment of α7 nicotinic acetylcholine receptor availability in juvenile pig brain with [18F]NS10743

    To conduct a quantitative PET assessment of the specific binding sites in the brain of juvenile pigs for [18F]NS10743, a novel diazabicyclononane derivative targeting α7 nicotinic acetylcholine receptors (α7 nAChRs). Dynamic PET recordings were made in isoflurane-anaesthetized juvenile pigs during 120 min after administration of [18F]NS10743 under baseline conditions (n = 3) and after blocking of the α7 nAChR with NS6740 (3 mg.kg-1 bolus + 1 mg.kg-1.h-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 [18F]NS10743 passed readily into the brain, with peak uptake occurring in α7 nAChR-expressing brain regions such as the colliculi, thalamus, temporal lobe and hippocampus. The highest SUVmax was approximately 2.3, whereas the lowest uptake was in the olfactory bulb (SUVmax 1.53 ± 0.32). Administration of NS6740 significantly decreased [18F]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 BPND. The baseline BPND 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 BPND in regions with high [18F]NS10743 binding (temporal lobe -29%, p = 0.01; midbrain: -35%, p = 0.02), without significantly altering the BPND in low binding regions (cerebellum: -16%, p = 0.2). This study confirms the potential of [18F]NS10743 as a target-specific radiotracer for the molecular imaging of central α7 nAChRs by PET. (orig.)

  16. Gene expression of ionotropic glutamate receptor subunits in the tectofugal pathway of the pigeon.

    Atoji, Y

    2016-03-01

    The tectofugal pathway in birds consists of four stations, the retina, optic tectum, rotundal nucleus, and entopallium, and it conveys visual information via three ascending pathways. These pathways consist of retino-tectal, tecto-rotundal and rotundo-entopallial cells, all of which are glutamatergic. The present study examined the localization of ionotropic glutamate receptors (iGluRs) to identify the target areas of glutamatergic projections in the tectofugal pathway in pigeons. Nine subunits of iGluRs were analyzed using in situ hybridization as follows: AMPA receptors (GluA1, GluA2, GluA3, and GluA4), kainate receptors (GluK1, GluK2, and GluK4), and NMDA receptors (GluN1 and GluN2A). Hybridization signals of subunits showed various intensities in different cells. In the optic tectum, a strong to moderate expression was observed in layer 10 (GluA2, GluA3, GluK4, and GluN1) and layer 13 (GluA2, GluK4, GluN1, and GluN2A). The rotundal nucleus intensely expressed GluA3, GluA4, GluK1, and GluK4. In the entopallium, an intense to moderate expression of GluK1 and GluK4, and a moderate to weak expression of AMPA and NMDA receptors were observed. Furthermore, the parvocellular and magnocellular parts of the isthmic nuclei showed a strong expression of GluA2, GluA3, GluK4, and GluN1. The present findings demonstrate the expression of iGluRs in glutamatergic projection targets of the tectofugal pathway in birds and suggest a diversity of iGluRs in the transmission of visual information. PMID:26718600

  17. Nicotine ameliorates NMDA receptor antagonist-induced deficits in contextual fear conditioning through high-affinity nicotinic acetylcholine receptors in the hippocampus.

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

    2011-03-01

    NMDA glutamate receptors (NMDARs) and nicotinic acetylcholine receptors (nAChRs) are both involved in learning and synaptic plasticity. Increasing evidence suggests processes mediated by these receptors may interact to modulate learning; however, little is known about the neural substrates involved in these interactive processes. The present studies investigated the effects of nicotine on MK-801 hydrogen maleate (MK-801) and DL-2-Amino-5-phosphonovaleric acid (APV)-induced disruption of contextual fear conditioning in male C57BL/6J mice, using direct drug infusion and selective nAChR antagonists to define the brain regions and the nAChR subtypes involved. Mice treated with MK-801 showed a deficit in contextual fear conditioning that was ameliorated by nicotine. Direct drug infusion demonstrated that the NMDAR antagonists disrupted hippocampal function and that nicotine acted in the dorsal hippocampus to ameliorate the deficit in learning. The high-affinity nAChR antagonist Dihydro-β-erythroidine hydrobromide (DhβE) blocked the effects of nicotine on MK-801-induced deficits while the α7 nAChR antagonist methyllycaconitine citrate salt hydrate (MLA) did not. These results suggest that NMDARs and nAChRs may mediate similar hippocampal processes involved in contextual fear conditioning. Furthermore, these results may have implications for developing effective therapeutics for the cognitive deficits associated with schizophrenia because a large subset of patients with schizophrenia exhibit cognitive deficits that may be related to NMDAR dysfunction and smoke at much higher rates than the healthy population, which may be an attempt to ameliorate cognitive deficits. PMID:21167848

  18. Age-Related Differences in NMDA Receptor Subunits of Prenatally Methamphetamine-Exposed Male Rats

    Vrajová, M.; Schutová, B.; Klaschka, Jan; Štěpánková, H.; Řípová, D.; Šlamberová, R.

    2014-01-01

    Roč. 39, č. 11 (2014), s. 2040-2046. ISSN 0364-3190 Grant ostatní: GA ČR(CZ) GAP303/10/0580; Ministerstvo školství(CZ) CSM 7/CRP/2014; Univerzita Karlova(CZ) Prvouk P34; Univerzita Karlova(CZ) 260045/SVV/2014; Prague Psychiatric Center(CZ) MH CZ–DRO: 00023752 Institutional support: RVO:67985807 Keywords : methamphetamine * in-utero * NMDA receptor subunits * hippocampus Subject RIV: FH - Neurology Impact factor: 2.593, year: 2014

  19. Radioiodination of nicotine with specific activity high enough for mapping nicotinic acetylcholine receptors

    A novel radiochemical method is presented to synthesize 5-[123I/125I/131I]-DL-nicotine by radioiodination of 5-bromonicotine. Radioiodination of the precursur 5-DL-bromonicotine was achieved using a copper (I)-assisted nucleophilic exchange reaction in the presence of reducing agent. The reaction conditions were optimized by varying pH, concentration of Sn(II) salt, ascorbic acid, Cu(I)chloride and reaction temperature. After purification by high-performance liquid chromatography the radiochemical purity of the product exceeded 98%, with a radiochemical yield of 55% and a specific activity ≥5 GBq/μmol. Specific binding of the iodinated nicotine was demonstrated in rate brain by autoradiography. The radioactivity from the specific structures was displaced by an excess of non-radioactive nicotine (10-3 M) with KD and Bmax of 13.1±7.8 nM and 22±2.7 fmol/mg protein and unspecific binding of about 40%. The in vivo distribution of 5-[131I]iodonicotine was determined in 20 female Wistar rats at various time intervals of 15 s to 90 min post injection (p.i.) by well counting and autoradiography. Brain activity peaked within 0.5 min p.i., and then showed a biexponential washout. Initially, activity within the cerebral cortex exceeded that of the cerebellum by a factor of 1.5-2.0. It was also increased in the striatum and thalamus. However, as soon as 15 min p.i. activity was almost homogeneously distributed. In conclusion, synthesis of 5-iodo-DL-nicotine (labelled with 131I, 125I or 123I, respectively) with appropriately high specific activity for receptor studies was achieved and specific binding to nicotine receptors in rat brain was demonstrated; following intravenous injection, however, there is considerable unspecific binding, obviously due to highly flow-dependent tissue retention. (orig.)

  20. Complex control of GABA(A) receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Mulligan, Megan K; Wang, Xusheng; Adler, Adrienne L; Mozhui, Khyobeni; Lu, Lu; Williams, Robert W

    2012-01-01

    GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal), even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer 4-100 fold

  1. Complex control of GABA(A receptor subunit mRNA expression: variation, covariation, and genetic regulation.

    Megan K Mulligan

    Full Text Available GABA type-A receptors are essential for fast inhibitory neurotransmission and are critical in brain function. Surprisingly, expression of receptor subunits is highly variable among individuals, but the cause and impact of this fluctuation remains unknown. We have studied sources of variation for all 19 receptor subunits using massive expression data sets collected across multiple brain regions and platforms in mice and humans. Expression of Gabra1, Gabra2, Gabrb2, Gabrb3, and Gabrg2 is highly variable and heritable among the large cohort of BXD strains derived from crosses of fully sequenced parents--C57BL/6J and DBA/2J. Genetic control of these subunits is complex and highly dependent on tissue and mRNA region. Remarkably, this high variation is generally not linked to phenotypic differences. The single exception is Gabrb3, a locus that is linked to anxiety. We identified upstream genetic loci that influence subunit expression, including three unlinked regions of chromosome 5 that modulate the expression of nine subunits in hippocampus, and that are also associated with multiple phenotypes. Candidate genes within these loci include, Naaa, Nos1, and Zkscan1. We confirmed a high level of coexpression for subunits comprising the major channel--Gabra1, Gabrb2, and Gabrg2--and identified conserved members of this expression network in mice and humans. Gucy1a3, Gucy1b3, and Lis1 are novel and conserved associates of multiple subunits that are involved in inhibitory signaling. Finally, proximal and distal regions of the 3' UTRs of single subunits have remarkably independent expression patterns in both species. However, corresponding regions of different subunits often show congruent genetic control and coexpression (proximal-to-proximal or distal-to-distal, even in the absence of sequence homology. Our findings identify novel sources of variation that modulate subunit expression and highlight the extraordinary capacity of biological networks to buffer

  2. Varenicline: a selective alpha4beta2 nicotinic acetylcholine receptor partial agonist approved for smoking cessation.

    Lam, Sum; Patel, Priti N

    2007-01-01

    Tobacco smoking remains a significant health problem in the United States. It has been associated with staggering morbidity and mortality, specifically due to malignancies and cardiovascular disease. Smoking cessation can be difficult and frequently requires pharmacologic interventions in addition to nonpharmacologic measures. Previously available agents are nicotine replacement products and bupropion, which increased quit rates by about 2-fold compared with placebo. Varenicline is the first drug in a new class known as the selective alpha4beta2 nicotinic receptor partial agonists. In several randomized, double-blind, 52-week clinical trials involving healthy chronic smokers, varenicline demonstrated superiority to placebo and bupropion in terms of efficacy measures. Additionally, it improved tobacco withdrawal symptoms and reinforcing effects of smoking in relapsed patients. Patients should start therapy in combination with tobacco cessation counseling 1 week before quit date and continue the regimen for 12 weeks. The dose of varenicline should be titrated to minimize nausea. The recommended dosage is 0.5 mg once daily (QD) on days 1-3; titrate to 0.5 mg twice daily (BID) on days 4-7; and 1 mg BID starting on day 8. An additional 12-week maintenance therapy may be considered for those who achieve abstinence. The most common side effects are nausea (30%), insomnia (18%), headache (15%), abnormal dreams (13%), constipation (8%), and abdominal pain (7%). Overall, varenicline is a breakthrough in the management of tobacco addiction and has demonstrated good efficacy in motivated quitters. It also provides an option for smokers who cannot tolerate other pharmacologic interventions. PMID:17438382

  3. Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and Gαq signaling

    Jordan, Theresa; Li, Jinyuan; Jiang, Hongbin; DiMario, Joseph X.

    2003-01-01

    Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and Gαq. Both mAchR and Gαq are abundant in medial adductor (MA) and PM fibers, and mAchR and Gαq interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 express...

  4. Multiple thyrotropin β-subunit and thyrotropin receptor-related genes arose during vertebrate evolution.

    Gersende Maugars

    Full Text Available Thyroid-stimulating hormone (TSH is composed of a specific β subunit and an α subunit that is shared with the two pituitary gonadotropins. The three β subunits derive from a common ancestral gene through two genome duplications (1R and 2R that took place before the radiation of vertebrates. Analysis of genomic data from phylogenetically relevant species allowed us to identify an additional Tshβ subunit-related gene that was generated through 2R. This gene, named Tshβ2, present in cartilaginous fish, little skate and elephant shark, and in early lobe-finned fish, coelacanth and lungfish, was lost in ray-finned fish and tetrapods. The absence of a second type of TSH receptor (Tshr gene in these species suggests that both TSHs act through the same receptor. A novel Tshβ sister gene, named Tshβ3, was generated through the third genomic duplication (3R that occurred early in the teleost lineage. Tshβ3 is present in most teleost groups but was lostin tedraodontiforms. The 3R also generated a second Tshr, named Tshrb. Interestingly, the new Tshrb was translocated from its original chromosomic position after the emergence of eels and was then maintained in its new position. Tshrb was lost in tetraodontiforms and in ostariophysians including zebrafish although the latter species have two TSHs, suggesting that TSHRb may be dispensable. The tissue distribution of duplicated Tshβs and Tshrs was studied in the European eel. The endocrine thyrotropic function in the eel would be essentially mediated by the classical Tshβ and Tshra, which are mainly expressed in the pituitary and thyroid, respectively. Tshβ3 and Tshrb showed a similar distribution pattern in the brain, pituitary, ovary and adipose tissue, suggesting a possible paracrine/autocrine mode of action in these non-thyroidal tissues. Further studies will be needed to determine the binding specificity of the two receptors and how these two TSH systems are interrelated.

  5. Early postnatal switch in GABAA receptor α-subunits in the reticular thalamic nucleus.

    Pangratz-Fuehrer, Susanne; Sieghart, Werner; Rudolph, Uwe; Parada, Isabel; Huguenard, John R

    2016-03-01

    The GABAergic neurons of the thalamic reticular nucleus (nRt) provide the primary source of inhibition within the thalamus. Using physiology, pharmacology, and immunohistochemistry in mice, we characterized postsynaptic developmental changes in these inhibitory projection neurons. First, at postnatal days 3-5 (P3-5), inhibitory postsynaptic currents (IPSCs) decayed very slowly, followed by a biphasic developmental progression, becoming faster at P6-8 and then slower again at P9-11 before stabilizing in a mature form around P12. Second, the pharmacological profile of GABA(A) receptor (GABA(A)R)-mediated IPSCs differed between neonatal and mature nRt neurons, and this was accompanied by reciprocal changes in α3 (late) and α5 (early) subunit expression in nRt. Zolpidem, selective for α1- and α3-containing GABA(A)Rs, augmented only mature IPSCs, whereas clonazepam enhanced IPSCs at all stages. This effect was blocked by the α5-specific inverse agonist L-655,708, but only in immature neurons. In α3(H126R) mice, in which α3-subunits were mutated to become benzodiazepine insensitive, IPSCs were enhanced compared with those in wild-type animals in early development. Third, tonic GABA(A)R activation in nRt is age dependent and more prominent in immature neurons, which correlates with early expression of α5-containing GABA(A)Rs. Thus neonatal nRt neurons show relatively high expression of α5-subunits, which contributes to both slow synaptic and tonic extrasynaptic inhibition. The postnatal switch in GABA(A)R subunits from α5 to α3 could facilitate spontaneous network activity in nRt that occurs at this developmental time point and which is proposed to play a role in early circuit development. PMID:26631150

  6. Deletion of the NMDA-NR1 receptor subunit gene in the mouse nucleus accumbens attenuates apomorphine-induced dopamine D1 receptor trafficking and acoustic startle behavior

    Glass, Michael J.; Robinson, Danielle C.; Waters, Elizabeth; Pickel, Virginia M.

    2013-01-01

    The nucleus accumbens (Acb) contains subpopulations of neurons defined by their receptor content and potential involvement in sensorimotor gating and other behaviors that are dysfunctional in schizophrenia. In Acb neurons, the NMDA NR1 (NR1) subunit is co-expressed not only with the dopamine D1 receptor (D1R), but also with the μ-opioid receptor (μ-OR), which mediates certain behaviors that are adversely impacted by schizophrenia. The NMDA-NR1 subunit has been suggested to play a role in the ...

  7. Conserved Expression of the Glutamate NMDA Receptor 1 Subunit Splice Variants during the Development of the Siberian Hamster Suprachiasmatic Nucleus

    Duffield, Giles E.; Jens D Mikkelsen; Ebling, Francis J. P.

    2012-01-01

    Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional propertie...

  8. Effects of Exposure to Aluminum on Long-term Potentiation and AMPA Receptor Subunits in Ratsin vivo

    SONG Jing; LIU Ying; ZHANG Hui Fang; ZHANG Qin Li; NIU Qiao

    2014-01-01

    ObjectiveTo explore the effects of exposure to aluminum(Al) on long-term potentiation(LTP) and AMPA receptor subunits in rats in vivo. MethodsDifferent dosages of aluminum-maltolate complex[Al(mal)3] were given to rats via acute intracerebroventricular (i.c.v.)injection and subchronic intraperitoneal (i.p.) injection. Following Al exposure, the hippocampal LTP were recorded by field potentiation techniquein vivo and the expression of AMPAR subunit proteins (GluR1 and GluR2) in both total and membrane-enriched extracts from the CA1 area of rat hippocampus were detected by Western blot assay. ResultsAcute Al treatment produced dose-dependent suppression of LTP in the rat hippocampus and dose-dependent decreases of GluR1and GluR2in membrane extracts; however, no similar changes were found in the total cell extracts, which suggests decreased trafficking of AMPA receptor subunits from intracellular pools to synaptic sites in the hippocampus. Thedose-dependent suppressive effects on LTP and the expression of AMPA receptor subunits both in the membrane and in total extracts were found after subchronic Al treatment, indicating a decrease in AMPA receptor subunit trafficking from intracellular poolsto synaptic sites and an additional reduction in the expression of the subunits. ConclusionAl(mal)3obviously and dose-dependently suppressed LTP in the rat hippocampal CA1 region in vivo, and this suppression may be related to both trafficking and decreases in the expression of AMPA receptor subunit proteins. However, the mechanisms underlying these observations need further investigation.

  9. Assessment of {alpha}7 nicotinic acetylcholine receptor availability in juvenile pig brain with [{sup 18}F]NS10743

    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.)

  10. Trypanosoma cruzi infection induces up-regulation of cardiac muscarinic acetylcholine receptors in vivo and in vitro

    K. Peraza-Cruces

    2008-09-01

    Full Text Available The pathogenesis of chagasic cardiomyopathy is not completely understood, but it has been correlated with parasympathetic denervation (neurogenic theory and inflammatory activity (immunogenic theory that could affect heart muscarinic acetylcholine receptor (mAChR expression. In order to further understand whether neurogenic and/or immunogenic alterations are related to changes in mAChR expression, we studied two models of Trypanosoma cruzi infection: 1 in 3-week-old male Sprague Dawley rats chronically infected with T. cruzi and 2 isolated primary cardiomyocytes co-cultured with T. cruzi and peripheral blood mononuclear cells (PBMC. Using [³H]-quinuclidinylbenzilate ([³H]-QNB binding assays, we evaluated mAChR expression in homogenates from selected cardiac regions, PBMC, and cultured cardiomyocytes. We also determined in vitro protein expression and pro-inflammatory cytokine expression in serum and cell culture medium by ELISA. Our results showed that: 1 mAChR were significantly (P < 0.05 up-regulated in right ventricular myocardium (means ± SEM; control: 58.69 ± 5.54, N = 29; Chagas: 72.29 ± 5.79 fmol/mg, N = 34 and PBMC (control: 12.88 ± 2.45, N = 18; Chagas: 20.22 ± 1.82 fmol/mg, N = 19, as well as in cardiomyocyte transmembranes cultured with either PBMC/T. cruzi co-cultures (control: 24.33 ± 3.83; Chagas: 43.62 ± 5.08 fmol/mg, N = 7 for both or their conditioned medium (control: 37.84 ± 3.84, N = 4; Chagas: 54.38 ± 6.28 fmol/mg, N = 20; 2 [³H]-leucine uptake was increased in cardiomyocytes co-cultured with PBMC/T. cruzi-conditioned medium (Chagas: 21,030 ± 2321; control 10,940 ± 2385 dpm, N = 7 for both; P < 0.05; 3 plasma IL-6 was increased in chagasic rats, IL-1β, was increased in both plasma of chagasic rats and in the culture medium, and TNF-α level was decreased in the culture medium. In conclusion, our results suggest that cytokines are involved in the up-regulation of mAChR in chronic Chagas disease.

  11. Effect of α{sub 7} nicotinic acetylcholine receptor agonists and antagonists on motor function in mice

    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.

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

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

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation...... recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine......,-non-selective inhibitors 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...

  13. Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively

    Collins, C; Duff, C; Duncan, A M;

    1993-01-01

    A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor...... subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1 to...... human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy that the...

  14. Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides.

    Bodereau-Dubois, Béatrice; List, Olivier; Calas-List, Delphine; Marques, Olivier; Communal, Pierre-Yves; Thany, Steeve H; Lapied, Bruno

    2012-05-01

    Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two α-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, ω-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential γ (TRPγ) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPγ function and nAChR2 sensitivity to acetamiprid. Similar TRPγ-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating

  15. Modulation of NR1 Subunit of N-Methyl-D-Aspartate Receptor by Ovariectomy and Passive Avoidance Learning

    Mahnaz Taherianfard

    2012-10-01

    Full Text Available BACKGROUND: Learning and memory are the most intensively studied subjects in neuroscience. Various approaches have been used to understand the underlying cellular and molecular mechanisms. Numerous studies have shown that different sites of brain are involved in learning and memory mechanisms. Two sites of mammalian brain that show high density of these receptors are CA1 region of hippocampus and Purkinje cell layer of cerebellum.METHODS: Twenty four Sprague-Dawley rats were used in 4 groups: Control-1 (intact without learning; control-2 (intact with learning; ovariectomy (OVX without learning and OVX with learning. A shuttle box apparatus used for passive avoidance learning procedure. Immunohistochemical procedure was used for determination of NR1 subunit of N-methyl-D-aspartate (NMDA receptor. Photoshop software was used for determination of color intensity.RESULTS: Immunohistological finding of this experiment indicated that OVX has a negative effect on density of NR1 subunit of NMDA receptors in two brain regions. Other finding of this study showed that passive avoidance learning significantly increased density of NR1 subunit of NMDA receptors in two brain regions.CONCLUSION: These results indicated that the sex hormone can modulate function and expression of the NR1 subunit of NMDA receptor in CA1 region of hippocampus and Purkinje cell layer of cerebellum.

  16. Acetylcholine receptor antibody

    ... Fenichel GM, Jankovic J, Mazziotta JC, eds. Bradley's Neurology in Clinical Practice . 6th ed. Philadelphia, PA: Elsevier ... 1/2015 Updated by: Daniel Kantor, MD, Kantor Neurology, Coconut Creek, FL and immediate past president of ...

  17. Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors.

    Yan, Feng; Mosier, Philip D; Westkaemper, Richard B; Roth, Bryan L

    2008-02-12

    Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Galpha-subunits (Galpha16 or Galphai2) with the kappa-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S3107.33, F3147.37, and I3167.39 to Y3207.43) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S3117.34 and N3267.49 were found to be sensitive in Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR. PMID:18205395

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

    Ekaterina N Lyukmanova

    Full Text Available SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1 differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM of human oral keratinocytes (Het-1A cells. Application of mecamylamine and atropine,--non-selective inhibitors 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-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM. It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1 did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the

  19. Expression of the GABA(A) receptor alpha6 subunit in cultured cerebellar granule cells is developmentally regulated by activation of GABA(A) receptors

    Carlson, B X; Belhage, B; Hansen, G H;

    1997-01-01

    Primary cultures of cerebellar granule cells, prepared from cerebella of 7-day-old rats and cultured for 4 or 8 days, were used to study the neurodifferentiative effect of a GABA(A) receptor agonist, 4,5,6,7-tetrahydroisoxazol[5,4-c]pyridin-3-ol (THIP), on the expression of the alpha6 GABA...... suggest that THIP has a trophic effect on alpha6 subunit expression, and this effect occurs only at an early developmental stage. Moreover, this study presents further evidence for the role of GABA(A) agonists, and thus the neurotransmitter, GABA, in regulating the expression of GABA(A) receptor subunits...

  20. Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

    Lanuza, M A; Li, M X; Jia, M; Kim, S; Davenport, R; Dunlap, V; Nelson, P G

    2000-09-15

    Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)-mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time- and dose-related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite-associated receptor aggregates in nerve-muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity-dependent synapse reduction and does so, at least in part, by altering AChR stability. PMID:10972958

  1. GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia

    LU, JUNJIE; ZHANG, QIAN; TAN, DONGMEI; LUO, WENPING; ZHAO, HAI; MA, JING; LIANG, HAO; TAN, YI

    2016-01-01

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE. PMID:27221053

  2. GABA A receptor π subunit promotes apoptosis of HTR-8/SVneo trophoblastic cells: Implications in preeclampsia.

    Lu, Junjie; Zhang, Qian; Tan, Dongmei; Luo, Wenping; Zhao, Hai; Ma, Jing; Liang, Hao; Tan, Yi

    2016-07-01

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter through its receptors in the mature central nervous system. The GABA type A receptor π subunit (GABRP) has been identified in the tissues of the reproductive system, particularly in the uterus. In addition, we have previously detected GABRP expression in both human and mouse placentas. To examine the role of GABRP in trophoblastic cell invasion, we constructed a pIRES2-GABRP-EGFP plasmid which was used for the transfection of a human placental cell line derived from first trimester extravillous trophoblasts (HTR-8/SVneo). The number of invaded cells was decreased by GABRP overexpression. Notably, the decrease in the invasive cell number may be due to the increased apoptosis of the HTR-8/SVneo cells following GABRP transfection, which was further confirmed by flow cytometry, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Based on the increased apoptosis of trophoblastic cells in pregnancies complicated by preeclampsia (PE) and the fact that GABRP promotes the apoptosis of trophoblastic cells, we hypothesized that GABRP expression is increased in the placental tissues from patients with PE compared with that in the normal groups and this hypothesis was confirmed by RT-qPCR and immunohistochemical analysis. Taken together, these findings imply that GABRP plays an important role in placentation and this pathway may be a promising molecular target for the development of novel therapeutic strategies for PE. PMID:27221053

  3. Resequencing of the auxiliary GABAB receptor subunit gene KCTD12 in chronic tinnitus

    Philipp G Sand

    2012-05-01

    Full Text Available Tinnitus is a common and often incapacitating hearing disorder marked by the perception of phantom sounds. Susceptibility factors remain largely unknown but GABAB receptor signalling has long been implicated in the response to treatment and, putatively, in the etiology of the disorder. We hypothesized that variation in KCTD12, the gene encoding an auxiliary subunit of GABAB receptors, could help to predict the risk of developing tinnitus. 95 Caucasian outpatients with a diagnosis of chronic tinnitus were systematically screened for mutations in the KCTD12 open reading frame and the adjacent 3' untranslated region by Sanger sequencing. Allele frequencies were determined for 14 known variants of which three (rs73237446, rs34544607 and rs41287030 were polymorphic. When allele frequencies were compared to data from a large reference population of European ancestry, rs34544607 was associated with tinnitus (p=.04. However, KCTD12 genotype did not predict tinnitus severity (p=.52 and the association with rs34544607 was weakened after screening 50 additional cases (p=.07. Pending replication in a larger cohort, KCTD12 may act as a risk modifier in chronic tinnitus. Issues that are yet to be addressed include the effects of neighbouring variants, e.g. in the KCTD12 gene regulatory region, plus interactions with variants of GABAB1 and GABAB2.

  4. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

    Amanda Lorraine Wright

    2012-04-01

    Full Text Available AMPA receptors are comprised of different combinations of GluR1-GluR4 (also known as GluA1-GluA4 and GluR-A to GluR-D subunits. The GluR2 subunit is subject to Q/R site RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Q, present in the GluR2 gene, to a codon for arginine (R found in the mRNA. AMPA receptors are calcium (Ca2+-permeable if they contain the unedited GluR2(Q subunit or if they lack the GluR2 subunit. While most AMPA receptors in the brain contain the edited GluR2(R subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable GluR2-lacking AMPA receptors are important in synaptic plasticity and learning. However, the presence of Ca2+-permeable AMPA receptors containing unedited GluR2 leads to excitotoxic cell loss. Recent studies have indicated that RNA editing of GluR2 is deregulated in diseases, such as amyotrophic lateral sclerosis (ALS, as well in acute neurodegenerative conditions, such as ischemia. More recently, studies have investigated the regulation of RNA editing and possible causes for its deregulation during disease. In this review, we will explore the role of GluR2 RNA editing in the healthy and diseased brain and outline new insights into the mechanisms that control this process.

  5. Novel α1 and γ2 GABAA receptor subunit mutations in families with idiopathic generalized epilepsy.

    Lachance-Touchette, Pamela; Brown, Patricia; Meloche, Caroline; Kinirons, Peter; Lapointe, Line; Lacasse, Hélène; Lortie, Anne; Carmant, Lionel; Bedford, Fiona; Bowie, Derek; Cossette, Patrick

    2011-07-01

    Epilepsy is a heterogeneous neurological disease affecting approximately 50 million people worldwide. Genetic factors play an important role in both the onset and severity of the condition, with mutations in several ion-channel genes being implicated, including those encoding the GABA(A) receptor. Here, we evaluated the frequency of additional mutations in the GABA(A) receptor by direct sequencing of the complete open reading frame of the GABRA1 and GABRG2 genes from a cohort of French Canadian families with idiopathic generalized epilepsy (IGE). Using this approach, we have identified three novel mutations that were absent in over 400 control chromosomes. In GABRA1, two mutations were found, with the first being a 25-bp insertion that was associated with intron retention (i.e. K353delins18X) and the second corresponding to a single point mutation that replaced the aspartate 219 residue with an asparagine (i.e. D219N). Electrophysiological analysis revealed that K353delins18X and D219N altered GABA(A) receptor function by reducing the total surface expression of mature protein and/or by curtailing neurotransmitter effectiveness. Both defects would be expected to have a detrimental effect on inhibitory control of neuronal circuits. In contrast, the single point mutation identified in the GABRG2 gene, namely P83S, was indistinguishable from the wildtype subunit in terms of surface expression and functionality. This finding was all the more intriguing as the mutation exhibited a high degree of penetrance in three generations of one French Canadian family. Further experimentation will be required to understand how this mutation contributes to the occurrence of IGE in these individuals. PMID:21714819

  6. Muscarinic acetylcholine receptor subtype 4 is essential for cholinergic stimulation of duodenal bicarbonate secretion in mice - relationship to D cell/somatostatin.

    Takeuchi, K; Kita, K; Takahashi, K; Aihara, E; Hayashi, S

    2015-06-01

    We investigated the roles of muscarinic (M) acetylcholine receptor subtype in the cholinergic stimulation of duodenal HCO3(-) secretion using knockout (KO) mice. Wild-type and M1-M5 KO C57BL/6J mice were used. The duodenal mucosa was mounted on an Ussing chamber, and HCO3(-) secretion was measured at pH 7.0 using a pH-stat method in vitro. Carbachol (CCh) or other agents were added to the serosal side. CCh dose-dependently stimulated HCO3(-) secretion in wild-type mice, and this effect was completely inhibited in the presence of atropine. The HCO3(-) response to CCh in wild-type mice was also inhibited by pirenzepine (M1 antagonist), 4DAMP (M3 antagonist), and tropicamide (M4 antagonist), but not by methoctramine (M2 antagonist). CCh stimulated HCO3(-) secretion in M2 and M5 KO animals as effectively as in WT mice; however, this stimulatory effect was significantly attenuated in M1, M3, and M4 KO mice. The decrease observed in the CCh-stimulated HCO3(-) response in M4 KO mice was reversed by the co-application of CYN154806, a somatostatin receptor type 2 (SST2) antagonist. Octreotide (a somatostatin analogue) decreased the basal and CCh-stimulated secretion of HCO3(-) in wild-type mice. The co-localized expression of somatostatin and M4 receptors was confirmed immunohistologically in the duodenum. We concluded that the duodenal HCO3(-) response to CCh was directly mediated by M1/M3 receptors and indirectly modified by M4 receptors. The activation of M4 receptors was assumed to inhibit the release of somatostatin from D cells and potentiate the HCO3(-) response by removing the negative influence of somatostatin via the activation of SST2 receptors. PMID:26084221

  7. The role of protein kinase-G in the antidepressant-like response of sildenafil in combination with muscarinic acetylcholine receptor antagonism

    Liebenberg, Nico; Wegener, Gregers; Brink, Christiaan; Harvey, Brian

    2010-01-01

    Background Recently we reported on a novel antidepressant-like response following simultaneous administration of sildenafil (PDE5 inhibitor, thereby increasing cGMP levels), and atropine (muscarinic acetylcholine receptor antagonist) in the rat forced swim test (FST). It is unclear whether the...... antidepressant-like activity of sildenafil + atropine is mediated via the activation of PK-G, a downstream effector for cGMP, and whether this may target known pathways in antidepressant action. Purpose We investigated whether the antidepressant-like response of sildenafil ± atropine could be reversed by Rp-8-Br......-PET-cGMP, a PK-G inhibitor, and also whether a combination of 8-Br-cGMP (PK-G activator) ± atropine would likewise be active in the FST, and whether this combination could be attenuated by a PK-G inhibitor. Study methods The FST consisted of a 15 minute pre-conditioning swim session on the first day of the...

  8. Guidelines for pre-clinical assessment of the acetylcholine receptor--specific passive transfer myasthenia gravis model-Recommendations for methods and experimental designs.

    Kusner, Linda L; Losen, Mario; Vincent, Angela; Lindstrom, Jon; Tzartos, Socrates; Lazaridis, Konstantinos; Martinez-Martinez, Pilar

    2015-08-01

    Antibodies against the muscle acetylcholine receptor (AChR) are the most common cause of myasthenia gravis (MG). Passive transfer of AChR antibodies from MG patients into animals reproduces key features of human disease, including antigenic modulation of the AChR, complement-mediated damage of the neuromuscular junction, and muscle weakness. Similarly, AChR antibodies generated by active immunization in experimental autoimmune MG models can subsequently be passively transferred to other animals and induce weakness. The passive transfer model is useful to test therapeutic strategies aimed at the effector mechanism of the autoantibodies. Here we summarize published and unpublished experience using the AChR passive transfer MG model in mice, rats and rhesus monkeys, and give recommendations for the design of preclinical studies in order to facilitate translation of positive and negative results to improve MG therapies. PMID:25743217

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

    Søderman, Andreas; Thomsen, Morten S; Hansen, Henrik 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...... that clinical trials testing alpha7 nAChR agonists should be related to the content of Abeta peptides in the patient's nervous system....... systemic administration of the alpha7 nAChR agonist SSR180711 (10 mg/kg) result in a significant increase in Fos protein levels in the shell of nucleus accumbens in wild-type mice, but has no effect in the transgene mice. There were fewer cell bodies expressing Fos in the prefrontal cortex of transgene...

  10. Decreased cerebral α4β2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer's disease assessed with positron emission tomography

    Postmortem studies indicate a loss of nicotinic acetylcholine receptor (nAChRs) in Alzheimer's disease (AD). In order to establish whether these changes in the cholinergic system occur at an early stage of AD, we carried out positron emission tomography (PET) with a specific radioligand for the α4β2* nicotinic acetylcholine receptor (α4β2* nAChR) in patients with mild to moderate AD and in patients with amnestic mild cognitive impairment (MCI), who have a high risk to progress to AD. Nine patients with moderate AD, eight patients with MCI and seven age-matched healthy controls underwent 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[18F]FA-85380) PET. After coregistration with individual magnetic resonance imaging the binding potential (BPND) of 2-[18F]FA-85380 was calculated using either the corpus callosum or the cerebellum as reference regions. PET data were analysed by region of interest analysis and by voxel-based analysis. Both patients with AD and MCI showed a significant reduction in 2-[18F]FA-85380 BPND in typical AD-affected brain regions. Thereby, the corpus callosum was identified as the most suitable reference region. The 2-[18F]FA-85380 BPND correlated with the severity of cognitive impairment. Only MCI patients that converted to AD in the later course (n = 5) had a reduction in 2-[18F]FA-85380 BPND. 2-[18F]FA-85380 PET appears to be a sensitive and feasible tool for the detection of a reduction in α4β2* nAChRs which seems to be an early event in AD. In addition, 2-[18F]FA-85380 PET might give prognostic information about a conversion from MCI to AD. (orig.)

  11. Decreased cerebral {alpha}4{beta}2* nicotinic acetylcholine receptor availability in patients with mild cognitive impairment and Alzheimer's disease assessed with positron emission tomography

    Kendziorra, Kai; Meyer, Philipp Mael; Barthel, Henryk; Hesse, Swen; Becker, Georg Alexander; Luthardt, Julia; Schildan, Andreas; Patt, Marianne; Sorger, Dietlind; Seese, Anita; Sabri, Osama [University of Leipzig, Department of Nuclear Medicine, Leipzig (Germany); Wolf, Henrike [University of Leipzig, Department of Psychiatry, Leipzig (Germany); University of Zurich, Department of Old Age Psychiatry and Psychiatry Research, Psychiatric University Hospital (PUK) Zurich, Zurich (Switzerland); Gertz, Herman-Josef [University of Leipzig, Department of Psychiatry, Leipzig (Germany)

    2011-03-15

    Postmortem studies indicate a loss of nicotinic acetylcholine receptor (nAChRs) in Alzheimer's disease (AD). In order to establish whether these changes in the cholinergic system occur at an early stage of AD, we carried out positron emission tomography (PET) with a specific radioligand for the {alpha}4{beta}2* nicotinic acetylcholine receptor ({alpha}4{beta}2* nAChR) in patients with mild to moderate AD and in patients with amnestic mild cognitive impairment (MCI), who have a high risk to progress to AD. Nine patients with moderate AD, eight patients with MCI and seven age-matched healthy controls underwent 2-[{sup 18}F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[{sup 18}F]FA-85380) PET. After coregistration with individual magnetic resonance imaging the binding potential (BP{sub ND}) of 2-[{sup 18}F]FA-85380 was calculated using either the corpus callosum or the cerebellum as reference regions. PET data were analysed by region of interest analysis and by voxel-based analysis. Both patients with AD and MCI showed a significant reduction in 2-[{sup 18}F]FA-85380 BP{sub ND} in typical AD-affected brain regions. Thereby, the corpus callosum was identified as the most suitable reference region. The 2-[{sup 18}F]FA-85380 BP{sub ND} correlated with the severity of cognitive impairment. Only MCI patients that converted to AD in the later course (n = 5) had a reduction in 2-[{sup 18}F]FA-85380 BP{sub ND}. 2-[{sup 18}F]FA-85380 PET appears to be a sensitive and feasible tool for the detection of a reduction in {alpha}4{beta}2* nAChRs which seems to be an early event in AD. In addition, 2-[{sup 18}F]FA-85380 PET might give prognostic information about a conversion from MCI to AD. (orig.)

  12. Evidence for cross-talk between M2 and M3 muscarinic acetylcholine receptors in the regulation of second messenger and extracellular signal-regulated kinase signalling pathways in Chinese hamster ovary cells

    Hornigold, David C; Mistry, Rajendra; Raymond, Pamela D; Blank, Jonathan L; John Challiss, R A

    2003-01-01

    We have examined possible mechanisms of cross-talk between the Gq/11-linked M3 muscarinic acetylcholine (mACh) receptor and the Gi/o-linked M2 mACh receptor by stable receptor coexpression in Chinese hamster ovary (CHO) cells. A number of second messenger (cyclic AMP, Ins(1,4,5)P3) and mitogen-activated protein kinase (ERK and JNK) responses stimulated by the mACh receptor agonist methacholine were examined in CHO-m2m3 cells and compared to those stimulated in CHO-m2 and CHO-m3 cell-lines, ex...

  13. Analysis of β-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives.

    Khom, S; Hintersteiner, J; Luger, D; Haider, M; Pototschnig, G; Mihovilovic, M D; Schwarzer, C; Hering, S

    2016-06-01

    Valerenic acid (VA)-a β2/3-selective GABA type A (GABAA) receptor modulator-displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed β-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1β1γ2S, α1β2γ2S, or α1β3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. β2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1β3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P tetrazole (α1β3γ2S: VA-TET: EC50 = 6.0 ± 1.0 μM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the β2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg/kg), whereas impairment of locomotion was observed at doses ≥10 mg/kg. PMID:27190170

  14. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    Lentes, K.U.; Venter, J.C.

    1986-05-01

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

  15. Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

    Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 μM clonazepam) with 10 nM 3H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 μg trypsin/mg membrane protein yielded H2O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain

  16. Targeting the γ-Aminobutyric Acid A Receptor α4 Subunit in Airway Smooth Muscle to Alleviate Bronchoconstriction.

    Yocum, Gene T; Gallos, George; Zhang, Yi; Jahan, Rajwana; Stephen, Michael Rajesh; Varagic, Zdravko; Puthenkalam, Roshan; Ernst, Margot; Cook, James M; Emala, Charles W

    2016-04-01

    We previously demonstrated that airway smooth muscle (ASM) cells express γ-aminobutyric acid A receptors (GABAARs), and that GABAAR agonists acutely relax ASM. Among the GABAAR α subunits, human ASM cells express only α4 and α5, providing the opportunity for selective pharmacologic targeting. Novel GABAAR-positive allosteric modulators designed for enhanced α4/α6 subunit selectivity were synthesized using iterative computational analyses (CMD-45 and XHe-III-74). Studies using oocyte heterologous expression systems confirmed that CMD-45 and XHe-III-74 led to significantly greater augmentation of currents induced by a 3% maximal effective concentration (EC3) of GABA [EC3]-induced currents in oocytes expressing α4 or α6 subunits (along with β3 and γ2) compared with other α subunits. CMD-45 and XHe-III-74 also led to greater ex vivo relaxation of contracted wild-type mouse tracheal rings compared with tracheal rings from GABAAR α4 subunit (Gabra4) knockout mice. Furthermore, CMD-45 and XHe-III-74 significantly relaxed precontracted human ASM ex vivo, and, at a low concentration, both ligands led to a significant leftward shift in albuterol-mediated ASM relaxation. In vivo, inhaled XHe-III-74 reduced respiratory system resistance in an asthmatic mouse model. Pretreatment of human ASM cells with CMD-45 and XHe-III-74 inhibited histamine-induced increases in intracellular calcium concentrations in vitro, an effect that was lost when calcium was omitted from the extracellular buffer, suggesting that inhibition of calcium influx due to alterations in plasma membrane potential may play a role in the mechanism of ASM relaxation. Selective targeting of the GABAAR α4 subunit with inhaled ligands may be a novel therapeutic pathway to treat bronchoconstriction, while avoiding sedative central nervous system effects, which are largely mediated by α1-3 subunit-containing GABAARs in the brain. PMID:26405827

  17. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-d-aspartate receptor subunits and d-serine ☆

    Savignac, Helene M.; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P.E.; Tzortzis, George; Burnet, Philip W. J.

    2013-01-01

    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we exami...

  18. Ovarian cycle-linked plasticity of δ-GABAA receptor subunits in hippocampal interneurons affects γ oscillations in vivo

    Albert Miklos Barth; Isabella eFerando; Istvan eMody

    2014-01-01

    GABAA receptors containing δ subunits (δ-GABAARs) are GABA-gated ion channels with extra- and perisynaptic localization, strong sensitivity to neurosteroids (NS), and a high degree of plasticity. In selective brain regions they are expressed on specific principal cells and interneurons (INs), and generate a tonic conductance that controls neuronal excitability and oscillations. Plasticity of δ-GABAARs in principal cells has been described during states of altered NS synthesis including acute...

  19. Expression of the Hippocampal NMDA Receptor GluN1 Subunit and Its Splicing Isoforms in Schizophrenia: Postmortem Study

    Vrajová, M.; Šťastný, František; Horáček, J.; Lochman, J.; Šerý, O.; Peková, S.; Klaschka, Jan; Höschl, C.

    2010-01-01

    Roč. 35, č. 7 (2010), s. 994-1002. ISSN 0364-3190 Grant ostatní: GA MZd(CZ) NR9324 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z10300504 Keywords : schizophrenia * hippocampus * GluN1 subunit of NMDA receptor * splice variants * laterality Subject RIV: FL - Psychiatry, Sexuology Impact factor: 2.608, year: 2010

  20. Breaking Tolerance in Transgenic Mice Expressing the Human TSH Receptor A-Subunit: Thyroiditis, Epitope Spreading and Adjuvant as a ‘Double Edged Sword’

    McLachlan, Sandra M.; Aliesky, Holly A.; Chen, Chun-Rong; Chong, Gao; Rapoport, Basil

    2012-01-01

    Transgenic mice with the human thyrotropin-receptor (TSHR) A-subunit targeted to the thyroid are tolerant of the transgene. In transgenics that express low A-subunit levels (Lo-expressors), regulatory T cell (Treg) depletion using anti-CD25 before immunization with adenovirus encoding the A-subunit (A-sub-Ad) breaks tolerance, inducing extensive thyroid lymphocytic infiltration, thyroid damage and antibody spreading to other thyroid proteins. In contrast, no thyroiditis develops in Hi-express...

  1. Neutralization og negative charges in F-loop of nicotinic acetylcholine alfa3beta4 receptors impairs the action of agonists and slows receptor desensitization

    Lindovský, Jiří; Kaniaková, Martina; Krůšek, Jan; Vyskočil, František

    Geneva : Swiss Society for Neuroscience, 2008. s. 212-212. ISBN 92-990014-3-X. [FENS. Forum of European Neuroscience /6./. 12.07.2008-16.07.2008, Geneva] R&D Projects: GA MŠk(CZ) LC554; GA AV ČR(CZ) IAA100110501; GA AV ČR(CZ) IAA5011411 Institutional research plan: CEZ:AV0Z50110509 Keywords : spo2 * acetylcholine * nicotine Subject RIV: ED - Physiology

  2. Effect of acetylcholine receptors on the pain-related electrical activities in the hippocampal CA3 region of morphine-addicted rats

    Guan Zeng Li

    2015-07-01

    Full Text Available 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 the sciatic nerve were used as noxious stimulation and the evoked electrical activities of PEN or PIN in hippocampal CA3 area were recorded using extracellular electrophysiological recording techniques in hippocampal slices. The effect of acetylcholine receptor stimulation byACh, the muscarinic agonist pilocarpine, and the muscarinic antagonist atropine on the pain evoked responses of pain related electrical activities was analyzed in hippocampal CA3 area of morphine addicted rats. Results:Intra-CA3 microinjection of ACh (2 μg/1 μl or pilocarpine (2 μg/1 μl decreased the discharge frequency and prolonged the firing latency of PEN, but increased the discharge frequency and shortened the firing inhibitory duration (ID of PIN. The intra-CA3 administration of atropine (0.5 μg/1 μl produced opposite effect. The peak activity of cholinergic modulators was 2 to 4 min later in morphine addicted rats compared to peak activity previously observed in normal rats. Conclusion: ACh dependent modulation of noxious stimulation exists in hippocampal CA3 area of morphine addicted rats. Morphine treatment may shift the sensitivity of pain related neurons towards a delayed response to muscarinergic neurotransmission in hippocampal CA3 region.

  3. A fungal metabolite asperparaline a strongly and selectively blocks insect nicotinic acetylcholine receptors: the first report on the mode of action.

    Koichi Hirata

    Full Text Available Asperparalines produced by Aspergillus japonicus JV-23 induce paralysis in silkworm (Bombyx mori larvae, but the target underlying insect toxicity remains unknown. In the present study, we have investigated the actions of asperparaline A on ligand-gated ion channels expressed in cultured larval brain neurons of the silkworm using patch-clamp electrophysiology. Bath-application of asperparaline A (10 µM had no effect on the membrane current, but when delivered for 1 min prior to co-application with 10 µM acetylcholine (ACh, it blocked completely the ACh-induced current that was sensitive to mecamylamine, a nicotinic acetylcholine receptor (nAChR-selective antaogonist. In contrast, 10 µM asperparaline A was ineffective on the γ-aminobutyric acid- and L-glutamate-induced responses of the Bombyx larval neurons. The fungal alkaloid showed no-use dependency in blocking the ACh-induced response with distinct affinity for the peak and slowly-desensitizing current amplitudes of the response to 10 µM ACh in terms of IC(50 values of 20.2 and 39.6 nM, respectively. Asperparaline A (100 nM reduced the maximum neuron response to ACh with a minimal shift in EC(50, suggesting that the alkaloid is non-competitive with ACh. In contrast to showing marked blocking action on the insect nAChRs, it exhibited only a weak blocking action on chicken α3β4, α4β2 and α7 nAChRs expressed in Xenopus laevis oocytes, suggesting a high selectivity for insect over certain vertebrate nAChRs.

  4. Presynaptic alpha-7 nicotinic acetylcholine receptors modulate excitatory synaptic transmission in hippocampal neurons%突触前α7烟碱受体对海马神经元兴奋性突触传递的调控

    刘振伟; 杨胜; 张永祥; 刘传缋

    2003-01-01

    The effects of presynaptic nicotinic acetylcholine receptors (nAChRs) on excitatory synaptic transmission in CA1 pyramidal neurons of the rat hippocampus were examined by blind whole-cell patch clamp recording from hippocampal slice preparations. Local application of the nAChRs agonist dimethylphenyl-piperazinium iodide (DMPP) did not induce a postsynaptic current response in CA1 pyramidal cells. However, DMPP enhanced the frequency and amplitude of spontaneous excitatory postsynaptic current (sEPSC) in these cells in a dose-dependent manner. This enhancement was blocked by the selective nicotinic α-7 receptor antagonist α-bungarotoxin, but not by the antagonist mecamylamine, hexamethonium or dihyhro3-erythroidine. The frequency of miniature excitatory postsynaptic current (mEPSC) in CA1 pyramidal neurons was also increased by application of DMPP, indicating a presynaptic site of action of the agonist. Taken together, these results suggest that activation of presynaptic nAChRs in CA1 pyramidal neurons, which contain α-7 subunits, potentiates presynaptic glutamate release and consequently modulate excitatory synaptic transmission in the hippocampus.%采用盲法膜片钳技术观察突触前烟碱受体(nicotinic acetylcholine receptors,nAChRs)对海马脑片CA1区锥体神经元兴奋性突触传递的调控作用.结果显示,nAChRs激动剂碘化二甲基苯基哌嗪(dimethylphenyl-piperazinium iodide,DMPP)不能在CA1区锥体神经元上诱发出烟碱电流.DMPP对CA1区锥体神经元自发兴奋性突触后电流(spontaneous excitatory postsynaptic current,sEPSC)具有明显的增频和增幅作用,并呈现明显的浓度依赖关系.DMPP对微小兴奋性突触后电流(miniature excitatory postsynaptic current,mEPSC)具有增频作用,但不具有增幅作用.上述DMPP增强突触传递的作用不能被nAChRs拮抗剂美加明、六烃季铵和双氢-β-刺桐丁所阻断,但可被α-银环蛇毒素阻断.上述结果提示,海马脑片CA1

  5. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

    2014-01-01

    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  6. Changes in brain striatum dopamine and acetylcholine receptors induced by chronic CDP-choline treatment of aging mice.

    Giménez, R.; Raïch, J.; Aguilar, J.

    1991-01-01

    1. Spiroperidol binding (dopamine D2 receptors) and quinuclidinyl benzilate binding (muscarinic receptors) in striata of 19-month old mice was analyzed for animals that had received chronic administration of cytidine 5'-diphosphocholine (CDP-choline) incorporated into the chow consumed (100 or 500 mg kg-1 added per day) for the 7 months before they were killed. 2. Treated animals displayed an increase in the dopamine receptor densities of 11% for those receiving 100 mg kg-1 and 18% for those ...

  7. [{sup 123}I]-3-Iodcytisin as possible radiotracer for the imaging of nicotinic acetylcholine receptors using single photon emission computer tomography; [{sup 123}I]-3-Iodcytisin als moeglicher Radiotracer fuer die Darstellung der nikotinergen Acetylcholin Rezeptoren mittels Single-Photon-Emissions-Computertomographie

    Paulik, Dagmar Julia

    2015-03-06

    For the synthesis of [{sup 123}I]-3-Iodcytisin as possible radiotracer for the imaging of nicotinic acetylcholine (nACh) receptors using SPECT two different technologies were used: the radio-iodination with iodogen and the radio-iodination with nitric acid. The latter one showed higher efficiency. The radiotracer will allow to detect degenerative processes and other nACh-depending diseases in the brain (Alzheimer, Parkinson) and to observe the progress. The autoradiography is aimed to the imaging of the nACh receptors in the brain bypassing the brain-blood barrier. The highest activity was measured in the thalamus of mice and rat brains.

  8. Antipsychotic-like effect of the muscarinic acetylcholine receptor agonist BuTAC in non-human primates

    Andersen, Maibritt B; Croy, Carrie Hughes; Dencker, Ditte;

    2015-01-01

    Cholinergic, muscarinic receptor agonists exhibit functional dopamine antagonism and muscarinic receptors have been suggested as possible future targets for the treatment of schizophrenia and drug abuse. The muscarinic ligand (5R,6R)-6-(3-butylthio-1,2,5-thiadiazol-4-yl)-1-azabicyclo[3.2.1]octane...

  9. Effect of developmental lead exposure on synaptic plasticity and N—methyl—D—aspartate receptor subunit in rat hippocampus

    RuanDY; SuiL

    2002-01-01

    Chronic lead(Pb) exposure is known to be associated with learning and memory,and cognitive dysfunction in children.Previous studies have demonstrated that Pb exposure may impair neuronal process underlying synaptic plasticity via a direct interaction with N-methyl-D-aspartate (NMDA) receptors(NMDARs).The studies described here were carried out to investigate effect of developmental Pb exposure on long-term potentiation(LTP),long-tern depression(LTD) and NMDAs subunits in rat hippocampus.The results are listed as follows:(1)low-level Pb exposture can impair the induction and maintenance of LTP in vivo and in vitro;(2)the Pb-induced impairment of LTD magnitude was an age-related decline in area CA1 of rat hippocampus;(3)chronic Pb exposure affected two components,voltage-gated calcium channel-dependent LTD and NMDARs-dependent LTD,of LTD induction in area CA1 of rat hippocampus;(4)different effects of developmental Pb exposure on NMDA receptor NR1,NR2A,NR2B,NR2C,NR2D and NR3A subunits in area CA1,CA2,CA3 and CA4 of rat hippocampus were observed;(5)the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors enriched in area CA1,CA3 and dentate gyrus and kainite receptors enriched in area CA1 and dentate gyrus of rat hippocampus were impaired by Pb exposure.

  10. Cocaine effects on mouse incentive-learning and human addiction are linked to α2 subunit-containing GABAA receptors

    Dixon, Claire I.; Morris, Hannah V.; Breen, Gerome; Desrivieres, Sylvane; Jugurnauth, Sarah; Steiner, Rebecca C.; Vallada, Homero; Guindalini, Camila; Laranjeira, Ronaldo; Messas, Guilherme; Rosahl, Thomas W.; Atack, John R.; Peden, Dianne R.; Belelli, Delia; Lambert, Jeremy J.; King, Sarah L.; Schumann, Gunter; Stephens, David N.

    2010-01-01

    Because GABAA receptors containing α2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with α2 gene deletion showed reduced synaptic GABAA receptor-mediated responses. Behaviorally, the deletion abolished cocaine’s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of α2-GABAA receptors (α2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In α2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of α2−GABAA receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction. PMID:20133874

  11. A Promising PET Tracer for Imaging of α7 Nicotinic Acetylcholine Receptors in the Brain: Design, Synthesis, and in Vivo Evaluation of a Dibenzothiophene-Based Radioligand

    Rodrigo Teodoro

    2015-10-01

    Full Text Available Changes in the expression of α7 nicotinic acetylcholine receptors (α7 nAChRs in the human brain are widely assumed to be associated with neurological and neurooncological processes. Investigation of these receptors in vivo depends on the availability of imaging agents such as radioactively labelled ligands applicable in positron emission tomography (PET. We report on a series of new ligands for α7 nAChRs designed by the combination of dibenzothiophene dioxide as a novel hydrogen bond acceptor functionality with diazabicyclononane as an established cationic center. To assess the structure-activity relationship (SAR of this new basic structure, we further modified the cationic center systematically by introduction of three different piperazine-based scaffolds. Based on in vitro binding affinity and selectivity, assessed by radioligand displacement studies at different rat and human nAChR subtypes and at the structurally related human 5-HT3 receptor, we selected the compound 7-(1,4-diazabicyclo[3.2.2]nonan-4-yl-2-fluorodibenzo-[b,d]thiophene 5,5-dioxide (10a for radiolabeling and further evaluation in vivo. Radiosynthesis of [18F]10a was optimized and transferred to an automated module. Dynamic PET imaging studies with [18F]10a in piglets and a monkey demonstrated high uptake of radioactivity in the brain, followed by washout and target-region specific accumulation under baseline conditions. Kinetic analysis of [18F]10a in pig was performed using a two-tissue compartment model with arterial-derived input function. Our initial evaluation revealed that the dibenzothiophene-based PET radioligand [18F]10a ([18F]DBT-10 has high potential to provide clinically relevant information about the expression and availability of α7 nAChR in the brain.

  12. Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

    Abongwa, Melanie; Buxton, Samuel K; Robertson, Alan P; Martin, Richard J

    2016-01-01

    Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex

  13. Curiouser and Curiouser: The Macrocyclic Lactone, Abamectin, Is also a Potent Inhibitor of Pyrantel/Tribendimidine Nicotinic Acetylcholine Receptors of Gastro-Intestinal Worms.

    Melanie Abongwa

    Full Text Available Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38, in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 μM abamectin in a non-competitive manner (reduced Rmax, no change in EC50. This antagonism increased when abamectin was increased to 0.1 μM. However, when we increased the concentration of abamectin further to 0.3 μM, 1 μM or 10 μM, we found that the antagonism decreased and was less than with 0.1 μM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM site causing antagonism, and another lower affinity positive allosteric (PAM site causing a reduction in antagonism. We also tested the effects of 0.1 μM derquantel alone and in combination with 0.3 μM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and

  14. Silencing gamma-aminobutyric acid A receptor alpha 1 subunit expression and outward potassium current in developing cortical neurons

    Tao Bo; Jiang Li; Jian Li; Xingfang Li; Kaihui Xing

    2011-01-01

    We used RNA interference (RNAi) to disrupt synthesis of the cortical neuronal γ-aminobutyric acid A receptor (GABAAR) α1 in rats during development, and measured outward K+ currents during neuronal electrical activity using whole-cell patch-clamp techniques. Three pairs of small interfering RNA (siRNA) for GABAAR α1 subunit were designed using OligoEngine RNAi software. This siRNA was found to effectively inhibited GABAAR α1 mRNA expression in cortical neuronal culture in vitro, but did not significantly affect neuronal survival. Outward K+ currents were decreased, indicating that GABAAR α1 subunits in developing neurons participate in neuronal function by regulating outward K+ current.

  15. Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons.

    Salgado, Vincent L; Saar, Raimund

    2004-10-01

    Two alpha-bungarotoxin-sensitive nicotinic receptor subtypes in cockroach neurons are identified as desensitizing (nAChD), selectively inhibitable with 100 nM imidacloprid, and non-desensitizing (nAChN), selectively inhibitable with 100 pM methyllycaconitine. Although the desensitization rate of nAChD receptors is highly variable, pharmacology is largely independent of desensitization rate. Because desensitized states tightly bind agonists, nAChD receptors are potently inhibited by neonicotinoids and specifically measured in radiolabeled imidacloprid binding assays. However, they are not usually detected in binding assays with radiolabeled alpha-bungarotoxin, which has a Kd for the resting state of 21 nM, but binds poorly to desensitized states often present in binding assays. In contrast, nAChN receptors are specifically measured in binding assays with radiolabeled alpha-bungarotoxin, which binds them with a Kd of 1.3 nM. nAChN receptors are activated by neonicotinoids at micromolar concentrations, and allosterically by spinosyn A, with an EC50 of 27 nM. Spinosyn A weakly antagonizes nAChD receptors -23% at 10 microM. The roles of the two nAChR subtypes in insecticide poisoning are discussed. PMID:15518655

  16. Mis-spliced transcripts of nicotinic acetylcholine receptor alpha6 are associated with field evolved spinosad resistance in Plutella xylostella (L..

    Simon W Baxter

    2010-01-01

    Full Text Available The evolution of insecticide resistance is a global constraint to agricultural production. Spinosad is a new, low-environmental-risk insecticide that primarily targets nicotinic acetylcholine receptors (nAChR and is effective against a wide range of pest species. However, after only a few years of application, field evolved resistance emerged in the diamondback moth, Plutella xylostella, an important pest of brassica crops worldwide. Spinosad resistance in a Hawaiian population results from a single incompletely recessive and autosomal gene, and here we use AFLP linkage mapping to identify the chromosome controlling resistance in a backcross family. Recombinational mapping with more than 700 backcross progeny positioned a putative spinosad target, nAChR alpha 6 (Pxalpha6, at the resistance locus, PxSpinR. A mutation within the ninth intron splice junction of Pxalpha6 results in mis-splicing of transcripts, which produce a predicted protein truncated between the third and fourth transmembrane domains. Additional resistance-associated Pxalpha6 transcripts that excluded the mutation containing exon were detected, and these were also predicted to produce truncated proteins. Identification of the locus of resistance in this important crop pest will facilitate field monitoring of the spread of resistance and offer insights into the genetic basis of spinosad resistance in other species.

  17. Exposure to nicotine increases nicotinic acetylcholine receptor density in the reward pathway and binge ethanol consumption in C57BL/6J adolescent female mice.

    Locker, Alicia R; Marks, Michael J; Kamens, Helen M; Klein, Laura Cousino

    2016-05-01

    Nearly 80% of adult smokers begin smoking during adolescence. Binge alcohol consumption is also common during adolescence. Past studies report that nicotine and ethanol activate dopamine neurons in the reward pathway and may increase synaptic levels of dopamine in the nucleus accumbens through nicotinic acetylcholine receptor (nAChR) stimulation. Activation of the reward pathway during adolescence through drug use may produce neural alterations affecting subsequent drug consumption. Consequently, the effect of nicotine exposure on binge alcohol consumption was examined along with an assessment of the neurobiological underpinnings that drive adolescent use of these drugs. Adolescent C57BL/6J mice (postnatal days 35-44) were exposed to either water or nicotine (200μg/ml) for ten days. On the final four days, ethanol intake was examined using the drinking-in-the-dark paradigm. Nicotine-exposed mice consumed significantly more ethanol and displayed higher blood ethanol concentrations than did control mice. Autoradiographic analysis of nAChR density revealed higher epibatidine binding in frontal cortical regions in mice exposed to nicotine and ethanol compared to mice exposed to ethanol only. These data show that nicotine exposure during adolescence increases subsequent binge ethanol consumption, and may affect the number of nAChRs in regions of the brain reward pathway, specifically the frontal cortex. PMID:26428091

  18. Nicotine promotes cell proliferation via α7-nicotinic acetylcholine receptor and catecholamine-synthesizing enzymes-mediated pathway in human colon adenocarcinoma HT-29 cells

    Cigarette smoking has been implicated in colon cancer. Nicotine is a major alkaloid in cigarette smoke. In the present study, we showed that nicotine stimulated HT-29 cell proliferation and adrenaline production in a dose-dependent manner. The stimulatory action of nicotine was reversed by atenolol and ICI 118,551, a β1- and β2-selective antagonist, respectively, suggesting the role of β-adrenoceptors in mediating the action. Nicotine also significantly upregulated the expression of the catecholamine-synthesizing enzymes [tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DβH) and phenylethanolamine N-methyltransferase]. Inhibitor of TH, a rate-limiting enzyme in the catecholamine-biosynthesis pathway, reduced the actions of nicotine on cell proliferation and adrenaline production. Expression of α7-nicotinic acetylcholine receptor (α7-nAChR) was demonstrated in HT-29 cells. Methyllycaconitine, an α7-nAChR antagonist, reversed the stimulatory actions of nicotine on cell proliferation, TH and DβH expression as well as adrenaline production. Taken together, through the action on α7-nAChR nicotine stimulates HT-29 cell proliferation via the upregulation of the catecholamine-synthesis pathway and ultimately adrenaline production and β-adrenergic activation. These data reveal the contributory role α7-nAChR and β-adrenoceptors in the tumorigenesis of colon cancer cells and partly elucidate the carcinogenic action of cigarette smoke on colon cancer

  19. Structural Analysis and Deletion Mutagenesis Define Regions of QUIVER/SLEEPLESS that Are Responsible for Interactions with Shaker-Type Potassium Channels and Nicotinic Acetylcholine Receptors.

    Meilin Wu

    Full Text Available Ly6 proteins are endogenous prototoxins found in most animals. They show striking structural and functional parallels to snake α-neurotoxins, including regulation of ion channels and cholinergic signaling. However, the structural contributions of Ly6 proteins to regulation of effector molecules is poorly understood. This question is particularly relevant to the Ly6 protein QUIVER/SLEEPLESS (QVR/SSS, which has previously been shown to suppress excitability and synaptic transmission by upregulating potassium (K channels and downregulating nicotinic acetylcholine receptors (nAChRs in wake-promoting neurons to facilitate sleep in Drosophila. Using deletion mutagenesis, co-immunoprecipitations, ion flux assays, surface labeling and confocal microscopy, we demonstrate that only loop 2 is required for many of the previously described properties of SSS in transfected cells, including interactions with K channels and nAChRs. Collectively our data suggest that QVR/SSS, and by extension perhaps other Ly6 proteins, target effector molecules using limited protein motifs. Mapping these motifs may be useful in rational design of drugs that mimic or suppress Ly6-effector interactions to modulate nervous system function.

  20. In vitro and ex vivo autoradiographic studies of nicotinic acetylcholine receptors using [18F]fluoronorchloroepibatidine in rodent and human brain

    A fluorine-18-labeled analog of the potent nicotinic agonist epibatidine is a candidate radioligand for positron emission tomographic (PET) studies of nicotinic acetylcholine receptors (nAcChR). Following intravenous administration of [18F]exo-2-(2'-fluoro-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane (NFEP), high uptake in thalamus was visualized in sections of mouse and rat brain by autoradiography using a phosphor imaging device. Binding of [18F]NFEP to rat thalamic homogenate was consistent with a single class of binding site with a Kd value of 71 pM. In vitro autoradiography of thaw-mounted sections of human thalamus revealed a heterogeneous pattern of binding; Bmax values for ventrolateral nucleus, insular cortex and dorsomedial nucleus, and internal capsule were 20, 8, and 3 pmol/cc of tissue, respectively. However, similar Kd values close to 50 pM were calculated for all regions. These studies support the suitability of [18F]NFEP as a radioligand for PET studies of nAcChR in the living human brain

  1. Transient Receptor Potential Channel Opening Releases Endogenous Acetylcholine, which Contributes to Endothelium-Dependent Relaxation Induced by Mild Hypothermia in Spontaneously Hypertensive Rat but Not Wistar-Kyoto Rat Arteries.

    Zou, Q; Leung, S W S; Vanhoutte, P M

    2015-08-01

    Mild hypothermia causes endothelium-dependent relaxations, which are reduced by the muscarinic receptor antagonist atropine. The present study investigated whether endothelial endogenous acetylcholine contributes to these relaxations. Aortic rings of spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were contracted with prostaglandin F2 α and exposed to progressive mild hypothermia (from 37 to 31°C). Hypothermia induced endothelium-dependent, Nω-nitro-l-arginine methyl ester-sensitive relaxations, which were reduced by atropine, but not by mecamylamine, in SHR but not in WKY rat aortae. The responses in SHR aortae were also reduced by acetylcholinesterase (the enzyme responsible for acetylcholine degradation), bromoacetylcholine (inhibitor of acetylcholine synthesis), hemicholinium-3 (inhibitor of choline uptake), and vesamicol (inhibitor of acetylcholine release). The mild hypothermia-induced relaxations in both SHR and WKY rat aortae were inhibited by AMTB [N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide; the transient receptor potential (TRP) M8 inhibitor]; only those in SHR aortae were inhibited by HC-067047 [2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide; TRPV4 antagonist] while those in WKY rat aortae were reduced by HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; TRPA1 antagonist]. The endothelial uptake of extracellular choline and release of cyclic guanosine monophosphate was enhanced by mild hypothermia and inhibited by HC-067047 in SHR but not in WKY rat aortae. Compared with WKY rats, the SHR preparations expressed similar levels of acetylcholinesterase and choline acetyltransferase, but a lesser amount of vesicular acetylcholine transporter, located mainly in the endothelium. Thus, mild hypothermia causes nitric oxide-dependent relaxations by opening TRPA1 channels in WKY rat aortae

  2. Bisphenol-A rapidly enhanced passive avoidance memory and phosphorylation of NMDA receptor subunits in hippocampus of young rats

    Bisphenol-A (BPA), an endocrine disruptor, is found to influence development of brain and behaviors in rodents. The previous study indicated that perinatal exposure to BPA impaired learning-memory and inhibited N-methyl-D-aspartate receptor (NMDAR) subunits expressions in hippocampus during the postnatal development in rats; and in cultured hippocampal neurons, BPA rapidly promotes dynamic changes in dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDAR subunit NR2B. In the present study, we examined the rapid effect of BPA on passive avoidance memory and NMDAR in the developing hippocampus of Sprague-Dawley rats at the age of postnatal day 18. The results showed that BPA or estradiol benzoate (EB) rapidly extended the latency to step down from the platform 1 h after footshock and increased the phosphorylation levels of NR1, NR2B, and mitogen-activated extracellular signal-regulated kinase (ERK) in hippocampus within 1 h. While 24 h after BPA or EB treatment, the improved memory and the increased phosphorylation levels of NR1, NR2B, ERK disappeared. Furthermore, pre-treatment with an estrogen receptors (ERs) antagonist, ICI182,780, or an ERK-activating kinase inhibitor, U0126, significantly attenuated EB- or BPA-induced phosphorylations of NR1, NR2B, and ERK within 1 h. These data suggest that BPA rapidly enhanced short-term passive avoidance memory in the developing rats. A non-genomic effect via ERs may mediate the modulation of the phosphorylation of NMDAR subunits NR1 and NR2B through ERK signaling pathway. - Highlights: → BPA rapidly extended the latency to step down from platform 1 h after footshock. → BPA rapidly increased pNR1, pNR2B, and pERK in hippocampus within 1 h. → ERs antagonist or MEK inhibitor attenuated BPA-induced pNR1, pNR2B, and pERK.

  3. Removal of GABA(A receptor γ2 subunits from parvalbumin neurons causes wide-ranging behavioral alterations.

    Elli Leppä

    Full Text Available We investigated the behavioral significance of fast synaptic inhibition by αβγ2-type GABA(A receptors on parvalbumin (Pv cells. The GABA(A receptor γ2 subunit gene was selectively inactivated in Pv-positive neurons by Cre/loxP recombination. The resulting Pv-Δγ2 mice were relatively healthy in the first postnatal weeks; but then as Cre started to be expressed, the mice progressively developed wide-ranging phenotypic alterations including low body weight, motor deficits and tremor, decreased anxiety levels, decreased pain sensitivity and deficient prepulse inhibition of the acoustic startle reflex and impaired spatial learning. Nevertheless, the deletion was not lethal, and mice did not show increased mortality even after one year. Autoradiography with t-butylbicyclophosphoro[(35S]thionate suggested an increased amount of GABA(A receptors with only α and β subunits in central nervous system regions that contained high levels of parvalbumin neurons. Using BAC-transgenesis, we reduced some of the Pv-Δγ2 phenotype by selectively re-expressing the wild-type γ2 subunit back into some Pv cells (reticular thalamic neurons and cerebellar Pv-positive neurons. This produced less severe impairments of motor skills and spatial learning compared with Pv-Δγ2 mice, but all other deficits remained. Our results reveal the widespread significance of fast GABAergic inhibition onto Pv-positive neurons for diverse behavioral modalities, such as motor coordination, sensorimotor integration, emotional behavior and nociception.

  4. Molecular target size of the vanilloid (capsaicin) receptor in pig dorsal root ganglia

    The size of the vanilloid receptor was examined by high-energy radiation inactivation analysis of the binding of [3H]resiniferatoxin to pig dorsal root ganglion membranes; it was found to be 270 ± 25 kDa. This value most likely represents the size of a receptor complex rather than of an individual subunit. Other ligand-gated cation channel complexes have reported molecular weights in this range, e.g. 300 kDa for the acetylcholine receptor

  5. Deletion of Asn{sup 281} in the {alpha}-subunit of the human insulin receptor causes constitutive activation of the receptor and insulin desensitization

    Desbois-Mouthon, C.; Sert-Langeron, C.; Magre, J.; Blivet, M.J. [INSERM, Paris (France)] [and others

    1996-02-01

    We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (<10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn{sup 281} in the {alpha}-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients` cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn{sup 281} of the IR {alpha}-subunit plays a critical role in the inhibitory constraint exerted by the extracellular {alpha}-subunit over the intracellular kinase activity. 59 refs., 6 figs.

  6. {alpha}4 {beta}2 nicotinic acetylcholine receptor in Alzheimer's disease and mild cognitive impairment: a study with 5-[I-123]iodo-A-85380 SPECT

    Park, Eun Kyung; Kim, Yu Kyeong; Kim, Sang Yun; Kim, Sang Eun [Seoul National Univ. College of Medicine, Seoul (Korea, Republic of)

    2007-07-01

    It has been reported that the number of neuronal nicotinic acetylcholine receptors (nAChRs) is decreased in Alzheimer's disease (AD) and the degree of cholinergic deficit is correlated with cognitive impairment. We examined neuronal nAChR distribution of AD patients using 5-[I-123]iodo-A85380 (5-IA) SPECT and correlated it with the pattern of cerebral glucose metabolic impairment and the severity of cognitive impairment. Five clinically diagnosed AD patients, 5 amnestic mild cognitive impairment (MCI) patients, and 10 age- and sex-matched healthy controls were studied with 5-IA SPECT and brain FDG PET. 5-IA SPECT was performed for 30 min at 120 min after radiotracer injection. FDG PET was done within one month interval. Neuropsychological tests were done for cognitive evaluation. A nAChR parameter DV was calculated in brain regions using cerebellum as reference tissue. All scan images were analyzed using SPM2 and ANOVA was done for group comparison. P value less than 0.005 was considered significant. 5-IA SPECT images of AD patients revealed significantly reduced nAChR distribution in the anterior cingulate cortex, striatum, and the left temporal cortex. MCI patients demonstrated decreased receptor distribution mainly in the subcortical areas. Cortical nAChR distribution showed correlation with cortical glucose metabolism and subcortical with that of posterior cingulate cortex (PCC). Episodic memory and semantic verbal fluency showed significant correlation with nAChR distribution of periventricular white matter (PVWM), visuospatial function evaluated with RCFT with that of PCC, left temporoparietal cortex, and frontal lobe white matter, and MMSE with that of PVWM, frontal cortex, and striatum. These data demonstrate reduction of nAChR distribution in patients with AD, which has significant correlation with cerebral glucose metabolism and cognitive impairment. It might be useful for diagnosis of AD, and for monitoring individualized treatments targeted at nAChRs.

  7. α4 β2 nicotinic acetylcholine receptor in Alzheimer's disease and mild cognitive impairment: a study with 5-[I-123]iodo-A-85380 SPECT

    It has been reported that the number of neuronal nicotinic acetylcholine receptors (nAChRs) is decreased in Alzheimer's disease (AD) and the degree of cholinergic deficit is correlated with cognitive impairment. We examined neuronal nAChR distribution of AD patients using 5-[I-123]iodo-A85380 (5-IA) SPECT and correlated it with the pattern of cerebral glucose metabolic impairment and the severity of cognitive impairment. Five clinically diagnosed AD patients, 5 amnestic mild cognitive impairment (MCI) patients, and 10 age- and sex-matched healthy controls were studied with 5-IA SPECT and brain FDG PET. 5-IA SPECT was performed for 30 min at 120 min after radiotracer injection. FDG PET was done within one month interval. Neuropsychological tests were done for cognitive evaluation. A nAChR parameter DV was calculated in brain regions using cerebellum as reference tissue. All scan images were analyzed using SPM2 and ANOVA was done for group comparison. P value less than 0.005 was considered significant. 5-IA SPECT images of AD patients revealed significantly reduced nAChR distribution in the anterior cingulate cortex, striatum, and the left temporal cortex. MCI patients demonstrated decreased receptor distribution mainly in the subcortical areas. Cortical nAChR distribution showed correlation with cortical glucose metabolism and subcortical with that of posterior cingulate cortex (PCC). Episodic memory and semantic verbal fluency showed significant correlation with nAChR distribution of periventricular white matter (PVWM), visuospatial function evaluated with RCFT with that of PCC, left temporoparietal cortex, and frontal lobe white matter, and MMSE with that of PVWM, frontal cortex, and striatum. These data demonstrate reduction of nAChR distribution in patients with AD, which has significant correlation with cerebral glucose metabolism and cognitive impairment. It might be useful for diagnosis of AD, and for monitoring individualized treatments targeted at nAChRs

  8. Rescue of amyloid-Beta-induced inhibition of nicotinic acetylcholine receptors by a peptide homologous to the nicotine binding domain of the alpha 7 subtype.

    Arthur A Nery

    Full Text Available Alzheimer's disease (AD is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs. Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

  9. Antidepressant activity in mice elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

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

    2014-05-21

    The objective of the current study is to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors (AChRs), produces antidepressant-like behavior in mice, and reactivates desensitized α7 AChRs expressed in CH3-α7 cells. Mice from both sexes were injected (i.p.) with PAM-2 (1.0mg/kg) on a daily basis for three weeks. Forced swim tests (FSTs) were performed on Day 1 and Day 7 to determine the acute and subchronic effects of PAM-2, respectively, and on Days 14 and 21 to determine its chronic activity. To examine the residual effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 28 and 35. Our results indicate that: (1) PAM-2 does not induce acute antidepressant effects in male or female mice, (2) PAM-2 induces antidepressant effects in mice from both sexes after one (subchronic) and two (chronic) weeks, whereas at the third week (chronic), the antidepressant effect is decreased in male and increased in female mice. Since PAM-2 does not influence the locomotor activity of mice, the observed antidepressant activity is not driven by nonspecific motor-stimulant actions, (3) the residual antidepressant effect mediated by PAM-2 after one week of treatment cessation is observed only in female mice, and finally the Ca(2+) influx results indicate that (4) PAM-2 can reactivate desensitized α7 AChRs. Our results clearly indicate that PAM-2 elicits antidepressant activity, probably by enhancing the activity of the endogenous neurotransmitter acetylcholine on α7 AChRs, without inducing receptor desensitization, and that this activity is gender-dependent. This is the first time that an antidepressant activity is described for an α7 PAM, supporting further studies as potential therapeutic medications for depressive states. PMID:24708923

  10. GABRR1 and GABRR2, encoding the GABA-A receptor subunits ρ1 and ρ2, are associated with alcohol dependence

    Xuei, Xiaoling; Flury-Wetherill, Leah; Dick, Danielle; Goate, Alison; Tischfield, Jay; Nurnberger, John; Schuckit, Marc; Kramer, John; Kuperman, Sam; Hesselbrock, Victor; Porjesz, Bernice; Foroud, Tatiana; Edenberg, Howard J.

    2010-01-01

    The genes encoding several GABA-A receptor subunits, including GABRA2, have been associated with alcoholism, suggesting that variations in GABA signaling contribute to risk. Therefore, as part of a comprehensive evaluation of the GABA receptor genes, we evaluated the potential association of GABRR1 and GABRR2, which encode the ρ1 and ρ2 subunits of the pentameric GABA-A/GABA-C receptors. GABRR1 and GABRR2 lie in a head to tail orientation spanning 137 kb on chromosome 6q14-16. We genotyped 73...

  11. Differential distribution of GABAA receptor subunits in soma and processes of cerebellar granule cells: effects of maturation and a GABA agonist

    Elster, L; Hansen, Gert Helge; Belhage, B;

    1995-01-01

    Quantitative analysis of the density of alpha 1 and beta 2/3 GABAA receptor subunits was performed at the electron microscope level after indirect pre-embedding immunogold labeling with subunit-specific antibodies of rat cerebellar granule cell cultures grown for 4 or 8 days and in the presence...... of receptors resulting in a single population of the latter neurons, a process enhanced by exposure to THIP. This may indicate that receptor development is a discontinuous process with individual neurons following different temporal patterns. In double-labeling experiments, a spatially close association...

  12. Effects of Rhizoma Acori Tatarinowii extracts on gamma-aminobutyric acid type A receptor alpha 1 subunit brain expression during development in a recurrent seizure rat model

    Liqun Liu; Ding'an Mao; Keqiang Chi; Xingfang Li; Tao Bo; Jinming Guo; Zhuwen Yi

    2011-01-01

    Extracts from Rhizoma Acori Tatarinowii (Grassleaf Sweetflag Rhizome, Shichangpu) have been shown to improve learning and memory, reduce anxiety, allay excitement, and suppress seizures. Rhizoma Acori Tatarinowii extracts interact with γ-aminobutyric acid and activate the γ-aminobutyric acid type A receptor, although few studies have addressed the precise effects of γ-aminobutyric acid type A receptor α1 subunit. In the present study, γ-aminobutyric acid type A receptor α1 subunit protein expression in the cerebral cortex and hippocampus, and pathological scores of brain injury, were significantly greater following recurrent seizures, but significantly decreased following treatment with Rhizoma Acori Tatarinowii extracts. These results indicated that Rhizoma Acori Tatarinowii extracts down-regulated γ-aminobutyric acid type A receptor α1 subunit protein expression in the cerebral cortex and hippocampus and protected seizure-induced brain injury during development.

  13. Short-term regulation of muscarinic acetylcholine receptors: An assessment utilizing mouse brain and mouse neuroblastoma cells

    Cioffi, C.L.

    1988-01-01

    The effects of muscarinic agonists and diisopropylfluorophosphate (DFP) on muscarinic receptor density and muscarinic receptor-mediated responses was assessed in mouse brain and mouse neuroblastoma cells (clone N1E-115). Utilizing the antagonist ({sup 3}H)quinuclidinyl benzilate (({sup 3}H)QNB), there was no difference in the maximal binding capacity (B{sub max}) or equilibrium dissociation constant (K{sub d}) between untreated and 24 hour DFP-treated mice. However, one administration of DFP produced a 24% and 33% decrease in B{sub max} measured by ({sup 3}H)N-methylscopolamine (({sup 3}H)NMS) after 18 and 24 hours which was rapidly reversible within 36 hours after DFP treatment. The loss of ({sup 3}H)NMS binding sites following acute DFP treatment was not accompanied by a change in a particular muscarinic receptor binding conformation. Furthermore, the magnitude of muscarinic receptor-mediated phosphoinositide hydrolysis was unchanged following short-term DFP treatment.

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

    Wang, Jing; Novak, Ivana

    2013-01-01

    , purinergic receptors, and determine their effects on ion transport. METHODS: Human adenocarcinoma cell line Capan-1 cells were grown on permeable supports and set in Ussing chambers for electrophysiological recordings. Transepithelial voltage (Vte), resistance, and short-circuit currents (Isc) were measured...

  15. Ric-3 chaperone-mediated stable cell-surface expression of the neuronal a7 nicotinic acetylcholine receptor in mammalian cells

    Ana Sofia VALLfiS; Ana M ROCCAMO; Francisco J BARRANTES

    2009-01-01

    Aim: Studies of the a7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of a7-type AChRs'11. The current work aims at obtaining and characterizing a cell line with high functional expression of the human a7 AChR.Methods: Ric-3 cDNA was incorporated into SHE-Pl-ha7 cells expressing the a7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [125I]a-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent a-bungarotoxin, anti-a7 antibody, and GFP-a7 were performed on the new clone.Results: The human a7-type AChR was stably expressed in a new cell line, which we coined SHE-PI-ha7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [125I]aBTX saturable binding with an apparent KD of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-PI-ha7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-a7 antibodies, hi contrast, chaperone-less SHE-PI-ha7 cells expressed only intracellular a.7 AChRs and failed to produce detectable single-channel currents.Conclusion: The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal a7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

  16. Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary.

    Townsend, Julie; Westcott, Karen; Tortonese, Domingo J

    2009-02-01

    In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common alpha-, LHbeta- and FSHbeta-subunit mRNA contents were determined by Northern analysis and the prevalence of LH-gonadotrophs assessed by immunohistochemistry in pituitaries from sexually active females (mares) in the BS, and sexually inactive mares in the NBS. These variables were then measured in castrated male horses (geldings). In mares, pituitary content of FSHbeta mRNA was significantly higher in the NBS (P0.05). These results reveal robust seasonal effects on common alpha-subunit and FSHbeta gene expression in the pituitary of the mare, in the absence of detectable changes in the content of LHbeta or GnRH-R mRNA. PMID:19114046

  17. The translational repressors Nanos and Pumilio have divergent effects on presynaptic terminal growth and postsynaptic glutamate receptor subunit composition.

    Menon, Kaushiki P; Andrews, Shane; Murthy, Mala; Gavis, Elizabeth R; Zinn, Kai

    2009-04-29

    Pumilio (Pum) is a translational repressor that binds selectively to target mRNAs and recruits Nanos (Nos) as a corepressor. In the larval neuromuscular system, Pum represses expression of the translation factor eIF-4E and the glutamate receptor subunit GluRIIA. Here, we show that Nos, like Pum, is expressed at the neuromuscular junction (NMJ) and in neuronal cell bodies. Surprisingly, however, Nos and Pum have divergent functions on both the presynaptic and postsynaptic sides of the NMJ. In nos mutant and nos RNA interference larvae, the number of NMJ boutons is increased, whereas loss of Pum reduces the bouton number. On the postsynaptic side, Nos acts in opposition to Pum in regulating the subunit composition of the glutamate receptor. NMJ active zones are associated with GluRIIA- and GluRIIB-containing receptor clusters. Loss of Nos causes downregulation of GluRIIA and increases the levels of GluRIIB. Consistent with this finding, the electrophysiological properties of NMJs lacking postsynaptic Nos suggest that they use primarily GluRIIB-containing receptors. Nos can regulate GluRIIB in the absence of GluRIIA, suggesting that the effects of Nos on GluRIIB levels are at least partially independent of synaptic competition between GluRIIA and GluRIIB. Nos is a target for Pum repression, and Pum binds selectively to the 3' untranslated regions of the nos and GluRIIA mRNAs. Our results suggest a model in which regulatory interplay among Pum, Nos, GluRIIA, and GluRIIB could cause a small change in Pum activity to be amplified into a large shift in the balance between GluRIIA and GluRIIB synapses. PMID:19403823

  18. Kalirin Binds the NR2B Subunit of the NMDA Receptor, Altering Its Synaptic Localization and Function

    Kiraly, D. D.

    2011-08-31

    The ability of dendritic spines to change size and shape rapidly is critical in modulating synaptic strength; these morphological changes are dependent upon rearrangements of the actin cytoskeleton. Kalirin-7 (Kal7), a Rho guanine nucleotide exchange factor localized to the postsynaptic density (PSD), modulates dendritic spine morphology in vitro and in vivo. Kal7 activates Rac and interacts with several PSD proteins, including PSD-95, DISC-1, AF-6, and Arf6. Mice genetically lacking Kal7 (Kal7KO) exhibit deficient hippocampal long-term potentiation (LTP) as well as behavioral abnormalities in models of addiction and learning. Purified PSDs from Kal7KO mice contain diminished levels of NR2B, an NMDA receptor subunit that plays a critical role in LTP induction. Here we demonstrate that Kal7KO animals have decreased levels of NR2B-dependent NMDA receptor currents in cortical pyramidal neurons as well as a specific deficit in cell surface expression of NR2B. Additionally, we demonstrate that the genotypic differences in conditioned place preference and passive avoidance learning seen in Kal7KO mice are abrogated when animals are treated with an NR2B-specific antagonist during conditioning. Finally, we identify a stable interaction between the pleckstrin homology domain of Kal7 and the juxtamembrane region of NR2B preceding its cytosolic C-terminal domain. Binding of NR2B to a protein that modulates the actin cytoskeleton is important, as NMDA receptors require actin integrity for synaptic localization and function. These studies demonstrate a novel and functionally important interaction between the NR2B subunit of the NMDA receptor and Kalirin, proteins known to be essential for normal synaptic plasticity.

  19. Prebiotic feeding elevates central brain derived neurotrophic factor, N-methyl-D-aspartate receptor subunits and D-serine.

    Savignac, Helene M; Corona, Giulia; Mills, Henrietta; Chen, Li; Spencer, Jeremy P E; Tzortzis, George; Burnet, Philip W J

    2013-12-01

    The influence of the gut microbiota on brain chemistry has been convincingly demonstrated in rodents. In the absence of gut bacteria, the central expression of brain derived neurotropic factor, (BDNF), and N-methyl-d-aspartate receptor (NMDAR) subunits are reduced, whereas, oral probiotics increase brain BDNF, and impart significant anxiolytic effects. We tested whether prebiotic compounds, which increase intrinsic enteric microbiota, also affected brain BDNF and NMDARs. In addition, we examined whether plasma from prebiotic treated rats released BDNF from human SH-SY5Y neuroblastoma cells, to provide an initial indication of mechanism of action. Rats were gavaged with fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS) or water for five weeks, prior to measurements of brain BDNF, NMDAR subunits and amino acids associated with glutamate neurotransmission (glutamate, glutamine, and serine and alanine enantiomers). Prebiotics increased hippocampal BDNF and NR1 subunit expression relative to controls. The intake of GOS also increased hippocampal NR2A subunits, and frontal cortex NR1 and d-serine. Prebiotics did not alter glutamate, glutamine, l-serine, l-alanine or d-alanine concentrations in the brain, though GOSfeeding raised plasma d-alanine. Elevated levels of plasma peptide YY (PYY) after GOS intake was observed. Plasma from GOS rats increased the release of BDNF from SH-SY5Y cells, but not in the presence of PYY antisera. The addition of synthetic PYY to SH-SY5Y cell cultures, also elevated BDNF secretion. We conclude that prebiotic-mediated proliferation of gut microbiota in rats, like probiotics, increases brain BDNF expression, possibly through the involvement of gut hormones. The effect of GOS on components of central NMDAR signalling was greater than FOS, and may reflect the proliferative potency of GOS on microbiota. Our data therefore, provide a sound basis to further investigate the utility of prebiotics in the maintenance of brain health and

  20. Placenta-specific Expression of the Interleukin-2 (IL-2) Receptor β Subunit from an Endogenous Retroviral Promoter*

    Cohen, Carla J.; Rebollo, Rita; Babovic, Sonja; Dai, Elizabeth L.; Robinson, Wendy P.; Mager, Dixie L.

    2011-01-01

    The long terminal repeat (LTR) sequences of endogenous retroviruses and retroelements contain promoter elements and are known to form chimeric transcripts with nearby cellular genes. Here we show that an LTR of the THE1D retroelement family has been domesticated as an alternative promoter of human IL2RB, the gene encoding the β subunit of the IL-2 receptor. The LTR promoter confers expression specifically in the placental trophoblast as opposed to its native transcription in the hematopoietic system. Rather than sequence-specific determinants, DNA methylation was found to regulate transcription initiation and splicing efficiency in a tissue-specific manner. Furthermore, we detected the cytoplasmic signaling domain of the IL-2Rβ protein in the placenta, suggesting that IL-2Rβ undergoes preferential proteolytic cleavage in this tissue. These findings implicate novel functions for this cytokine receptor subunit in the villous trophoblast and reveal an intriguing example of ancient LTR exaptation to drive tissue-specific gene expression. PMID:21865161