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Sample records for ach receptor achr

  1. Identification of phosphorylation sites on AChR delta-subunit associated with dispersal of AChR clusters on the surface of muscle cells.

    PubMed

    Nimnual, A S; Chang, W; Chang, N S; Ross, A F; Gelman, M S; Prives, J M

    1998-10-20

    The innervation of embryonic skeletal muscle cells is marked by the redistribution of nicotinic acetylcholine receptors (AChRs) on muscle surface membranes into high-density patches at nerve-muscle contacts. To investigate the role of protein phosphorylation pathways in the regulation of AChR surface distribution, we have identified the sites on AChR delta-subunits that undergo phosphorylation associated with AChR cluster dispersal in cultured myotubes. We found that PKC-catalyzed AChR phosphorylation is targeted to Ser378, Ser393, and Ser450, all located in the major intracellular domain of the AChR delta-subunit. Adjacent to one of these sites is a PKA consensus target site (Ser377) that was efficiently phosphorylated by purified PKA in vitro. The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and the phosphoprotein phosphatase inhibitor okadaic acid (OA) produced increased phosphorylation of AChR delta-subunits on the three serine residues that were phosphorylated by purified PKC in vitro. In contrast, treatment of these cells with the PKA activator forskolin, or with the cell-permeable cAMP analogue 8-bromo-cAMP, did not alter the phosphorylation state of surface AChR, suggesting that PKA does not actively phosphorylate the delta-subunit in intact chick myotubes. The effects of TPA and OA included an increase in the proportion of surface AChR that is extracted in Triton X-100, as well as the spreading of AChR from cluster regions to adjacent areas of the muscle cell surface. These findings suggest that PKC-catalyzed phosphorylation on the identified serine residues of AChR delta-subunits may play a role in the surface distribution of these receptors. PMID:9778356

  2. Acetylcholine Receptor (AChR) Clustering Is Regulated Both by Glycogen Synthase Kinase 3β (GSK3β)-dependent Phosphorylation and the Level of CLIP-associated Protein 2 (CLASP2) Mediating the Capture of Microtubule Plus-ends*

    PubMed Central

    Basu, Sreya; Sladecek, Stefan; Pemble, Hayley; Wittmann, Torsten; Slotman, Johan A.; van Cappellen, Wiggert; Brenner, Hans-Rudolf; Galjart, Niels

    2014-01-01

    The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2–9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2–8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends. PMID:25231989

  3. Myasthenia Gravis and the Tops and Bottoms of AChRs Antigenic Structure of the MIR and Specific Immunosuppression of EAMG Using AChR Cytoplasmic Domains

    PubMed Central

    Lindstrom, Jon; Luo, Jie; Kuryatov, Alexander

    2009-01-01

    The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of α1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR α3 subunits, but MG patient antibodies do not. By making chimeras of α1 subunits with α7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of α1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in α1/α7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human α1, β1, γ, δ, and ε subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG. PMID:18567851

  4. Salbutamol and ephedrine in the treatment of severe AChR deficiency syndromes

    PubMed Central

    Rodríguez Cruz, Pedro M.; Palace, Jacqueline; Ramjattan, Hayley; Jayawant, Sandeep; Robb, Stephanie A.

    2015-01-01

    Objective: To evaluate the response to salbutamol and ephedrine in the treatment of congenital myasthenic syndromes due to CHRNE mutations causing severe acetylcholine receptor (AChR) deficiency. Methods: A cohort study of 6 patients with severe AChR deficiency, symptomatic despite optimal therapy with anticholinesterase and 3,4-diaminopyridine, were analyzed for their response to the addition of salbutamol or ephedrine to their medication. Baseline quantitative myasthenia gravis (QMG) (severity) scores were worse than 15 of 39. Patients were assessed in clinic with QMG and mobility scores. Pretreatment and 6- to 8-month follow-up scores were evaluated. Results: All 6 patients tolerated treatment well and reported no side effects. There was a strong positive response to treatment over the 6- to 8-month assessment period with significant improvement in QMG (p = 0.027) and mobility scores. The analysis of subcomponents of the QMG score revealed marked improvement in upper (p = 0.028) and lower (p = 0.028) limb raise times. All patients reported enhanced activities of daily living at 6 to 8 months. Conclusions: Oral salbutamol and ephedrine appear to be effective treatments in severe cases of AChR deficiency on pyridostigmine. They are well tolerated and improvement in strength can be dramatic. Classification of evidence: This study provides Class IV evidence that salbutamol or ephedrine improves muscle strength in patients with congenital myasthenia from severe AChR deficiency. PMID:26296515

  5. Expression of human AChR extracellular domain mutants with improved characteristics.

    PubMed

    Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Giastas, Petros; Bitzopoulou, Kalliopi; Evangelakou, Panagiota; Sideri, Anastasia; Tzartos, Socrates J

    2014-02-01

    The muscle nicotinic acetylcholine receptor (AChR) has a central role in neuromuscular transmission, and is the major target in the autoimmune disease myasthenia gravis (MG). We created mutants of the extracellular domains (ECDs) of the human α1, β1, δ and ε AChR subunits, whereby their Cys-loop was exchanged for that of the acetylcholine binding protein. The mutants were expressed in Pichia pastoris and had improved solubility resulting in 2- to 43-fold higher expression yields compared to the wild type. An additional mutant was created for the α1 ECD restoring its glycosylation site within the Cys-loop and its α-bungarotoxin binding ability. Furthermore, we constructed dimeric and pentameric concatamers of the mutant ECDs. All concatamers were successfully expressed as soluble secreted proteins, although the pentamers had about 10-fold lower expression than the dimers and were more susceptible to fragmentation. Initial crystallizations with the mutant ECDs were promising, and we reproducibly obtained crystals of the β1 ECD, diffracting at ~12 Å. Further optimization is underway to obtain crystals suitable for high resolution crystallography. The proteins described herein are useful tools in structural studies of the human muscle AChR and can be used in applications requiring high yields such as therapeutic adsorbents for MG autoantibodies. PMID:24246999

  6. Efficient Expression of Functional (α6β2)2β3 AChRs in Xenopus Oocytes from Free Subunits Using Slightly Modified α6 Subunits

    PubMed Central

    Ley, Carson Kai-Kwong; Kuryatov, Alexander; Wang, Jingyi; Lindstrom, Jon Martin

    2014-01-01

    Human (α6β2)(α4β2)β3 nicotinic acetylcholine receptors (AChRs) are essential for addiction to nicotine and a target for drug development for smoking cessation. Expressing this complex AChR is difficult, but has been achieved using subunit concatamers. In order to determine what limits expression of α6* AChRs and to efficiently express α6* AChRs using free subunits, we investigated expression of the simpler (α6β2)2β3 AChR. The concatameric form of this AChR assembles well, but is transported to the cell surface inefficiently. Various chimeras of α6 with the closely related α3 subunit increased expression efficiency with free subunits and produced pharmacologically equivalent functional AChRs. A chimera in which the large cytoplasmic domain of α6 was replaced with that of α3 increased assembly with β2 subunits and transport of AChRs to the oocyte surface. Another chimera replacing the unique methionine 211 of α6 with leucine found at this position in transmembrane domain 1 of α3 and other α subunits increased assembly of mature subunits containing β3 subunits within oocytes. Combining both α3 sequences in an α6 chimera increased expression of functional (α6β2)2β3 AChRs to 12-fold more than with concatamers. This is pragmatically useful, and provides insights on features of α6 subunit structure that limit its expression in transfected cells. PMID:25068303

  7. Myopathic changes detected by quantitative electromyography in patients with MuSK and AChR positive myasthenia gravis.

    PubMed

    Nikolic, Ana; Basta, Ivana; Stojanovic, Vidosava Rakocevic; Stevic, Zorica; Peric, Stojan; Lavrnic, Dragana

    2016-05-01

    Myopathic changes are frequent a electrophysiological finding in patients with muscle specific tyrosine kinase (MuSK) positive myasthenia gravis (MG). The aim of this study was to explore the importance of quantitative electromyography (EMG) in the detection of myopathic changes in MuSK MG patients. Classical and quantitative EMG were performed in 31 MuSK and 28 acetylcholine receptor (AChR) positive MG patients, matched by sex, age, disease duration and severity. Classical EMG revealed the presence of myopathic changes more frequently in MuSK MG compared to AChR MG patients, especially in the facial muscles. Quantitative EMG registered myopathic lesions more frequently than classical EMG, but the frequency was similar between MuSK and AChR MG patients. Quantitative EMG revealed myopathic changes in the majority of both MuSK and AChR positive MG patients. This examination is sensitive, but it cannot be used to differentiate between MG patients belonging to the different disease groups. It should not be used in isolation. Rather, it should complement classical EMG in the detection of myopathic changes. PMID:26778359

  8. Direct Proof of the In Vivo Pathogenic Role of the AChR Autoantibodies from Myasthenia Gravis Patients

    PubMed Central

    Kordas, Gregory; Lagoumintzis, George; Sideris, Sotirios; Poulas, Konstantinos; Tzartos, Socrates J.

    2014-01-01

    Several studies have suggested that the autoantibodies (autoAbs) against muscle acetylcholine receptor (AChR) of myasthenia gravis (MG) patients are the main pathogenic factor in MG; however, this belief has not yet been confirmed with direct observations. Although animals immunized with AChR or injected with anti-AChR monoclonal Abs, or with crude human MG Ig fractions exhibit MG symptoms, the pathogenic role of isolated anti-AChR autoAbs, and, more importantly, the absence of pathogenic factor(s) in the autoAb-depleted MG sera has not yet been shown by in vivo studies. Using recombinant extracellular domains of the human AChR α and β subunits, we have isolated autoAbs from the sera of four MG patients. The ability of these isolated anti-subunit Abs and of the Ab-depleted sera to passively transfer experimental autoimmune MG in Lewis rats was investigated. We found that the isolated anti-subunit Abs were at least as efficient as the corresponding whole sera or whole Ig in causing experimental MG. Abs to both α- and β-subunit were pathogenic although the anti-α-subunit were much more efficient than the anti-β-subunit ones. Interestingly, the autoAb-depleted sera were free of pathogenic activity. The later suggests that the myasthenogenic potency of the studied anti-AChR MG sera is totally due to their anti-AChR autoAbs, and therefore selective elimination of the anti-AChR autoAbs from MG patients may be an efficient therapy for MG. PMID:25259739

  9. Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR.

    PubMed

    Mukhtasimova, Nuriya; daCosta, Corrie J B; Sine, Steven M

    2016-07-01

    The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist-receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate

  10. Mutations in GFPT1 that underlie limb-girdle congenital myasthenic syndrome result in reduced cell-surface expression of muscle AChR.

    PubMed

    Zoltowska, Katarzyna; Webster, Richard; Finlayson, Sarah; Maxwell, Susan; Cossins, Judith; Müller, Juliane; Lochmüller, Hanns; Beeson, David

    2013-07-15

    Mutations in GFPT1 underlie a congenital myasthenic syndrome (CMS) characterized by a limb-girdle pattern of muscle weakness. Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is a key rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for the glycosylation of proteins and lipids. It is expressed ubiquitously and it is not readily apparent why mutations in this gene should cause a syndrome with symptoms restricted to muscle and, in particular, to the neuromuscular junction. Data from a muscle biopsy obtained from a patient with GFPT1 mutations indicated that there were reduced endplate acetylcholine receptors. We, therefore, further investigated the relationship between identified mutations in GFPT1 and expression of the muscle acetylcholine receptor. Cultured myotubes derived from two patients with GFPT1 mutations showed a significant reduction in cell-surface AChR expression (Pt1 P < 0.0001; Pt2 P = 0.0097). Inhibition of GFPT1 enzymatic activity or siRNA silencing of GFPT1 expression both resulted in reduced AChR cell-surface expression. Western blot and gene-silencing experiments indicate this is due to reduced steady-state levels of AChR α, δ, ε, but not β subunits rather than altered transcription of AChR-subunit RNA. Uridine diphospho-N-acetylglucosamine, a product of the hexosamine synthetic pathway, acts as a substrate at an early stage in the N-linked glycosylation pathway. Similarity between CMS due to GFPT1 mutations and CMS due to DPAGT1 mutations would suggest that reduced endplate AChR due to defective N-linked glycosylation is a primary disease mechanism in this disorder. PMID:23569079

  11. Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

    PubMed Central

    Bourne, Yves; Radić, Zoran; Aráoz, Rómulo; Talley, Todd T.; Benoit, Evelyne; Servent, Denis; Taylor, Palmer; Molgó, Jordi; Marchot, Pascale

    2010-01-01

    Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type α12βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP (≈2.4Å) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity. PMID:20224036

  12. Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism.

    PubMed

    Bourne, Yves; Radic, Zoran; Aráoz, Rómulo; Talley, Todd T; Benoit, Evelyne; Servent, Denis; Taylor, Palmer; Molgó, Jordi; Marchot, Pascale

    2010-03-30

    Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type alpha1(2)betagammadelta and neuronal alpha3beta2 and alpha4beta2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP ( approximately 2.4A) show toxins neatly imbedded within the nest of ar-omatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bis-spiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the se-quence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity. PMID:20224036

  13. Muscle-specific kinase (MuSK) autoantibodies suppress the MuSK pathway and ACh receptor retention at the mouse neuromuscular junction

    PubMed Central

    Ghazanfari, Nazanin; Morsch, Marco; Reddel, Stephen W; Liang, Simon X; Phillips, William D

    2014-01-01

    Muscle-specific kinase (MuSK) autoantibodies from myasthenia gravis patients can block the activation of MuSK in vitro and/or reduce the postsynaptic localization of MuSK. Here we use a mouse model to examine the effects of MuSK autoantibodies upon some key components of the postsynaptic MuSK pathway and upon the regulation of junctional ACh receptor (AChR) numbers. Mice became weak after 14 daily injections of anti-MuSK-positive patient IgG. The intensity and area of AChR staining at the motor endplate was markedly reduced. Pulse-labelling of AChRs revealed an accelerated loss of pre-existing AChRs from postsynaptic AChR clusters without a compensatory increase in incorporation of (newly synthesized) replacement AChRs. Large, postsynaptic AChR clusters were replaced by a constellation of tiny AChR microaggregates. Puncta of AChR staining also appeared in the cytoplasm beneath the endplate. Endplate staining for MuSK, activated Src, rapsyn and AChR were all reduced in intensity. In the tibialis anterior muscle there was also evidence that phosphorylation of the AChR β-subunit-Y390 was reduced at endplates. In contrast, endplate staining for β-dystroglycan (through which rapsyn couples AChR to the synaptic basement membrane) remained intense. The results suggest that anti-MuSK IgG suppresses the endplate density of MuSK, thereby down-regulating MuSK signalling activity and the retention of junctional AChRs locally within the postsynaptic membrane scaffold. PMID:24860174

  14. miR-434-3p and DNA hypomethylation co-regulate eIF5A1 to increase AChRs and to improve plasticity in SCT rat skeletal muscle.

    PubMed

    Shang, Fei-Fei; Xia, Qing-Jie; Liu, Wei; Xia, Lei; Qian, Bao-Jiang; You, Ling; He, Mu; Yang, Jin-Liang; Wang, Ting-Hua

    2016-01-01

    Acetylcholine receptors (AChRs) serve as connections between motor neurons and skeletal muscle and are essential for recovery from spinal cord transection (SCT). Recently, microRNAs have emerged as important potential biotherapeutics for several diseases; however, whether miRNAs operate in the modulation of AChRs remains unknown. We found increased AChRs numbers and function scores in rats with SCT; these increases were reduced following the injection of a eukaryotic translation initiation factor 5A1 (eIF5A1) shRNA lentivirus into the hindlimb muscle. Then, high-throughput screening for microRNAs targeting eIF5A1 was performed, and miR-434-3p was found to be robustly depleted in SCT rat skeletal muscle. Furthermore, a highly conserved miR-434-3p binding site was identified within the mRNA encoding eIF5A1 through bioinformatics analysis and dual-luciferase assay. Overexpression or knockdown of miR-434-3p in vivo demonstrated it was a negative post-transcriptional regulator of eIF5A1 expression and influenced AChRs expression. The microarray-enriched Gene Ontology (GO) terms regulated by miR-434-3p were muscle development terms. Using a lentivirus, one functional gene (map2k6) was confirmed to have a similar function to that of miR-434-3p in GO terms. Finally, HRM and MeDIP-PCR analyses revealed that DNA demethylation also up-regulated eIF5A1 after SCT. Consequently, miR-434-3p/eIF5A1 in muscle is a promising potential biotherapy for SCI repair. PMID:26964899

  15. miR-434-3p and DNA hypomethylation co-regulate eIF5A1 to increase AChRs and to improve plasticity in SCT rat skeletal muscle

    PubMed Central

    Shang, Fei-Fei; Xia, Qing-Jie; Liu, Wei; Xia, Lei; Qian, Bao-Jiang; You, Ling; He, Mu; Yang, Jin-Liang; Wang, Ting-Hua

    2016-01-01

    Acetylcholine receptors (AChRs) serve as connections between motor neurons and skeletal muscle and are essential for recovery from spinal cord transection (SCT). Recently, microRNAs have emerged as important potential biotherapeutics for several diseases; however, whether miRNAs operate in the modulation of AChRs remains unknown. We found increased AChRs numbers and function scores in rats with SCT; these increases were reduced following the injection of a eukaryotic translation initiation factor 5A1 (eIF5A1) shRNA lentivirus into the hindlimb muscle. Then, high-throughput screening for microRNAs targeting eIF5A1 was performed, and miR-434-3p was found to be robustly depleted in SCT rat skeletal muscle. Furthermore, a highly conserved miR-434-3p binding site was identified within the mRNA encoding eIF5A1 through bioinformatics analysis and dual-luciferase assay. Overexpression or knockdown of miR-434-3p in vivo demonstrated it was a negative post-transcriptional regulator of eIF5A1 expression and influenced AChRs expression. The microarray-enriched Gene Ontology (GO) terms regulated by miR-434-3p were muscle development terms. Using a lentivirus, one functional gene (map2k6) was confirmed to have a similar function to that of miR-434-3p in GO terms. Finally, HRM and MeDIP-PCR analyses revealed that DNA demethylation also up-regulated eIF5A1 after SCT. Consequently, miR-434-3p/eIF5A1 in muscle is a promising potential biotherapy for SCI repair. PMID:26964899

  16. Differential Cytokine Changes in Patients with Myasthenia Gravis with Antibodies against AChR and MuSK

    PubMed Central

    Yilmaz, Vuslat; Oflazer, Piraye; Aysal, Fikret; Durmus, Hacer; Poulas, Kostas; Yentur, Sibel P.; Gulsen-Parman, Yesim; Tzartos, Socrates; Marx, Alexander; Tuzun, Erdem; Deymeer, Feza; Saruhan-Direskeneli, Güher

    2015-01-01

    Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-γ, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-γ and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients

  17. Differential Cytokine Changes in Patients with Myasthenia Gravis with Antibodies against AChR and MuSK.

    PubMed

    Yilmaz, Vuslat; Oflazer, Piraye; Aysal, Fikret; Durmus, Hacer; Poulas, Kostas; Yentur, Sibel P; Gulsen-Parman, Yesim; Tzartos, Socrates; Marx, Alexander; Tuzun, Erdem; Deymeer, Feza; Saruhan-Direskeneli, Güher

    2015-01-01

    Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-γ, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-γ and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients

  18. MuSK Myasthenia Gravis IgG4 Disrupts the Interaction of LRP4 with MuSK but Both IgG4 and IgG1-3 Can Disperse Preformed Agrin-Independent AChR Clusters

    PubMed Central

    Koneczny, Inga; Cossins, Judith; Waters, Patrick; Beeson, David; Vincent, Angela

    2013-01-01

    A variable proportion of patients with generalized myasthenia gravis (MG) have autoantibodies to muscle specific tyrosine kinase (MuSK). During development agrin, released from the motor nerve, interacts with low density lipoprotein receptor-related protein-4 (LRP4), which then binds to MuSK; MuSK interaction with the intracellular protein Dok7 results in clustering of the acetylcholine receptors (AChRs) on the postsynaptic membrane. In mature muscle, MuSK helps maintain the high density of AChRs at the neuromuscular junction. MuSK antibodies are mainly IgG4 subclass, which does not activate complement and can be monovalent, thus it is not clear how the antibodies cause disruption of AChR numbers or function to cause MG. We hypothesised that MuSK antibodies either reduce surface MuSK expression and/or inhibit the interaction with LRP4. We prepared MuSK IgG, monovalent Fab fragments, IgG1-3 and IgG4 fractions from MuSK-MG plasmas. We asked whether the antibodies caused endocytosis of MuSK in MuSK-transfected cells or if they inhibited binding of LRP4 to MuSK in co-immunoprecipitation experiments. In parallel, we investigated their ability to reduce AChR clusters in C2C12 myotubes induced by a) agrin, reflecting neuromuscular development, and b) by Dok7- overexpression, producing AChR clusters that more closely resemble the adult neuromuscular synapse. Total IgG, IgG4 or IgG1-3 MuSK antibodies were not endocytosed unless cross-linked by divalent anti-human IgG. MuSK IgG, Fab fragments and IgG4 inhibited the binding of LRP4 to MuSK and reduced agrin-induced AChR clustering in C2C12 cells. By contrast, IgG1-3 antibodies did not inhibit LRP4-MuSK binding but, surprisingly, did inhibit agrin-induced clustering. Moreover, both IgG4 and IgG1-3 preparations dispersed agrin-independent AChR clusters in Dok7-overexpressing C2C12 cells. Thus interference by IgG4 antibodies of the LRP4-MuSK interaction will be one pathogenic mechanism of MuSK antibodies, but IgG1-3 Mu

  19. Patient autoantibodies deplete postsynaptic muscle-specific kinase leading to disassembly of the ACh receptor scaffold and myasthenia gravis in mice.

    PubMed

    Cole, R N; Ghazanfari, N; Ngo, S T; Gervásio, O L; Reddel, S W; Phillips, W D

    2010-09-01

    The postsynaptic muscle-specific kinase (MuSK) coordinates formation of the neuromuscular junction (NMJ) during embryonic development. Here we have studied the effects of MuSK autoantibodies upon the NMJ in adult mice. Daily injections of IgG from four MuSK autoantibody-positive myasthenia gravis patients (MuSK IgG; 45 mg day(1)i.p. for 14 days) caused reductions in postsynaptic ACh receptor (AChR) packing as assessed by fluorescence resonance energy transfer (FRET). IgG from the patients with the highest titres of MuSK autoantibodies caused large (51-73%) reductions in postsynaptic MuSK staining (cf. control mice; P < 0.01) and muscle weakness. Among mice injected for 14 days with control and MuSK patient IgGs, the residual level of MuSK correlated with the degree of impairment of postsynaptic AChR packing. However, the loss of postsynaptic MuSK preceded this impairment of postsynaptic AChR. When added to cultured C2 muscle cells the MuSK autoantibodies caused tyrosine phosphorylation of MuSK and the AChR beta-subunit, and internalization of MuSK from the plasma membrane. The results suggest a pathogenic mechanism in which MuSK autoantibodies rapidly deplete MuSK from the postsynaptic membrane leading to progressive dispersal of postsynaptic AChRs. Moreover, maintenance of postsynaptic AChR packing at the adult NMJ would appear to depend upon physical engagement of MuSK with the AChR scaffold, notwithstanding activation of the MuSK-rapsyn system of AChR clustering. PMID:20603331

  20. The open duration of fetal ACh receptor-channel changes during mouse muscle development

    PubMed Central

    Grassi, Francesca; Epifano, Olga; Mileo, Anna Maria; Barabino, Benedetta; Eusebi, Fabrizio

    1998-01-01

    We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the γ subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5. We studied unitary ACh-evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6). At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24-26 °C. On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the γs subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period. PMID:9508804

  1. Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots

    PubMed Central

    Geng, Lin; Peng, H. Benjamin

    2014-01-01

    In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs. PMID:24587270

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

    PubMed

    Rotzler, S; Brenner, H R

    1990-08-01

    The effects of muscle activity on the growth of synaptic acetylcholine receptor (AChR) accumulations and on the metabolic AChR stability were investigated in rat skeletal muscle. Ectopic end plates induced surgically in adult soleus muscle were denervated early during development when junctional AChR number and stability were still low and, subsequently, muscles were either left inactive or they were kept active by chronic exogenous stimulation. AChR numbers per ectopic AChR cluster and AChR stabilities were estimated from the radioactivity and its decay with time, respectively, of end plate sites whose AChRs had been labeled with 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. PMID:2380246

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

    PubMed

    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

  4. Otilonium: a potent blocker of neuronal nicotinic ACh receptors in bovine chromaffin cells.

    PubMed Central

    Gandía, L.; Villarroya, M.; Lara, B.; Olmos, V.; Gilabert, J. A.; López, M. G.; Martínez-Sierra, R.; Borges, R.; García, A. G.

    1996-01-01

    1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by

  5. Clinical application of clustered-AChR for the detection of SNMG

    PubMed Central

    Zhao, Guang; Wang, Xiaoqing; Yu, Xiaowen; Zhang, Xiutian; Guan, Yangtai; Jiang, Jianming

    2015-01-01

    Myasthenia gravis (MG) is an autoantibody-mediated disease of the neuromuscular junction (NMJ). However, accumulating evidence has indicated that MG patients whose serum anti-acetylcholine receptor (AChR) antibodies are not detectable (serumnegative MG; SNMG) in routine assays share similar clinical features with anti-AChR antibody-positive MG patients. We hypothesized that SNMG patients would have low-affinity antibodies to AChRs that would not be detectable using traditional methods but that might be detected by binding to AChR on the cell membrane, particularly if they were clustered at the high density observed at the NMJ. We expressed AChR subunits with the clustering protein rapsyn (an AChR-associated protein at the synapse) in human embryonic kidney (HEK) cells, and we tested the binding of the antibodies using immunofluorescence. With this approach, AChR antibodies to rapsyn-clustered AChR could be detected in the sera from 45.83% (11/24) of SNMG patients, as confirmed with fluorescence-activated cell sorting (FACS). This was the first application in China of cell-based AChR antibody detection. More importantly, this sensitive (and specific) approach could significantly increase the diagnosis rate of SNMG. PMID:26068604

  6. Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody.

    PubMed

    Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

    2013-05-15

    In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

  7. Pyridostigmine but not 3,4-diaminopyridine exacerbates ACh receptor loss and myasthenia induced in mice by muscle-specific kinase autoantibody

    PubMed Central

    Morsch, Marco; Reddel, Stephen W; Ghazanfari, Nazanin; Toyka, Klaus V; Phillips, William D

    2013-01-01

    In myasthenia gravis, the neuromuscular junction is impaired by the antibody-mediated loss of postsynaptic acetylcholine receptors (AChRs). Muscle weakness can be improved upon treatment with pyridostigmine, a cholinesterase inhibitor, or with 3,4-diaminopyridine, which increases the release of ACh quanta. The clinical efficacy of pyridostigmine is in doubt for certain forms of myasthenia. Here we formally examined the effects of these compounds in the antibody-induced mouse model of anti-muscle-specific kinase (MuSK) myasthenia gravis. Mice received 14 daily injections of IgG from patients with anti-MuSK myasthenia gravis. This caused reductions in postsynaptic AChR densities and in endplate potential amplitudes. Systemic delivery of pyridostigmine at therapeutically relevant levels from days 7 to 14 exacerbated the anti-MuSK-induced structural alterations and functional impairment at motor endplates in the diaphragm muscle. No such effect of pyridostigmine was found in mice receiving control human IgG. Mice receiving smaller amounts of MuSK autoantibodies did not display overt weakness, but 9 days of pyridostigmine treatment precipitated generalised muscle weakness. In contrast, one week of treatment with 3,4-diaminopyridine enhanced neuromuscular transmission in the diaphragm muscle. Both pyridostigmine and 3,4-diaminopyridine increase ACh in the synaptic cleft yet only pyridostigmine potentiated the anti-MuSK-induced decline in endplate ACh receptor density. These results thus suggest that ongoing pyridostigmine treatment potentiates anti-MuSK-induced AChR loss by prolonging the activity of ACh in the synaptic cleft. PMID:23440963

  8. Involvement of calpains in the destabilization of the acetylcholine receptor clusters in rat myotubes.

    PubMed

    Kim, S; Nelson, P G

    2000-01-01

    The effects of calpain inhibitors on the total number of acetylcholine receptors (AChRs) on cultured rat myotubes and on the stability of AChR clusters in these myotubes were investigated. The degradation rate of total AChRs labeled with (125)I-alpha-bungarotoxin was assessed from radioactivity remaining in the myotubes as a function of time. Treatment with calpain inhibitors resulted in a two- to three-fold increase in the half-life of total AChRs. Incubation with these inhibitors produced 40% increases in intracellular AChRs but no major changes in surface AChRs, indicating that the increased AChR half-life is due to intracellular accumulation. The rate loss of AChRs from the clusters was assessed by measuring the loss of fluorescence intensity in rhodaminated-alpha-bungarotoxin-labeled clusters with time. Treatment with calpain inhibitors resulted in twofold increases in cluster half-life. Thus, there was generally no change in total surface receptors with the calpain inhibitors, whereas cluster half-life was substantially increased. Furthermore, with a low dose of calpeptin there was no change in turnover of total cellular AChRs, whereas cluster half-life was doubled. Taken together, these results suggest that the increased half-life of clusters produced by the calpain inhibitors may be due to retardation of the lateral movement from AChRs in the clusters. PMID:10623898

  9. Recycling of Acetylcholine Receptors at Ectopic Postsynaptic Clusters Induced by Exogenous Agrin in Living Rats

    PubMed Central

    Brenner, Hans Rudolf; Akaaboune, Mohammed

    2014-01-01

    During the development of the neuromuscular junction, motor axons induce the clustering of acetylcholine receptors (AChRs) and increase their metabolic stability in the muscle membrane. Here, we asked whether the synaptic organizer agrin might regulate the metabolic stability and density of AChRs by promoting the recycling of internalized AChRs, which would otherwise be destined for degradation, into synaptic sites. We show that at nerve-free AChR clusters induced by agrin in extrasynaptic membrane, internalized AChRs are driven back into the ectopic synaptic clusters where they intermingle with pre-existing and new receptors. The extent of AChR recycling depended on the strength of the agrin stimulus, but not on the development of junctional folds, another hallmark of mature postsynaptic membranes. In chronically denervated muscles, in which both AChR stability and recycling are significantly decreased by muscle inactivity, agrin maintained the amount of recycled AChRs at agrin-induced clusters at a level similar to that at denervated original endplates. In contrast, AChRs did not recycle at agrin-induced clusters in C2C12 or primary myotubes. Thus, in muscles in vivo, but not in cultured myotubes, neural agrin promotes the recycling of AChRs and thereby increases their metabolic stability. PMID:25093969

  10. Turnover of acetylcholine receptors: Mechanisms of regulation. Final report, 1 August 1985-30 November 1990

    SciTech Connect

    Drachman, D.B.

    1990-12-31

    The synthesis, insertion and degradation of acetylcholine receptors (AChRs) of skeletal muscle cells as closely regulated both by the muscle cells and by the motor nerves that supply them. The goal of this project is to elucidate the mechanisms of regulation of the AChRs, both at the neuromuscular junctional and at extrajunctional regions. The results of our studies on junctional AChRs have shown that: Both stable and rapidly turned over (RTO) AChRs are present at normally innervated neuromuscular junctions` Synthesis and insertion of AChRs at neuromuscular junctions occurs rapidly, at a rate consistent with the rapid rate of turnover of RTOs. RTOs serve as precursors of stable AChRs. Acetylcholine receptors, RA5 Neuromuscular junctions, Motor nerves.

  11. A Case Report of Congenital Fiber Type Disproportion with an Increased Level of Anti-ACh Receptor Antibodies.

    PubMed

    Kimura, Shigemi; Ozasa, Shiro; Nomura, Keiko; Kosuge, Hirofumi; Yoshioka, Kowasi

    2013-01-01

    Congenital fiber type disproportion (CFTD) is a form of congenital myopathy, which is defined by type 1 myofibers that are 12% smaller than type 2 myofibers, as well as a general predominance of type 1 myofibers. Conversely, myasthenia gravis (MG) is an acquired immune-mediated disease, in which the acetylcholine receptor (AChR) of the neuromuscular junction is blocked by antibodies. Thus, the anti-AChR antibody is nearly specific to MG. Herein, we report on a case of CFTD with increased anti-AChR antibody levels. A 23-month-old boy exhibited muscle hypotonia and weakness. Although he could walk by himself, he easily fell down and could not control his head for a long time. His blood test was positive for the anti-AChR antibody, while a muscle biopsy revealed characteristics of CFTD. We could not explain the relationship between MG and CFTD. However, we considered different diagnoses aside from MG, even when the patient's blood is positive for the anti-AChR antibody. PMID:23762716

  12. Muscarinic ACh Receptors Contribute to Aversive Olfactory Learning in Drosophila

    PubMed Central

    Silva, Bryon; Molina-Fernández, Claudia; Ugalde, María Beatriz; Tognarelli, Eduardo I.; Angel, Cristian; Campusano, Jorge M.

    2015-01-01

    The most studied form of associative learning in Drosophila consists in pairing an odorant, the conditioned stimulus (CS), with an unconditioned stimulus (US). The timely arrival of the CS and US information to a specific Drosophila brain association region, the mushroom bodies (MB), can induce new olfactory memories. Thus, the MB is considered a coincidence detector. It has been shown that olfactory information is conveyed to the MB through cholinergic inputs that activate acetylcholine (ACh) receptors, while the US is encoded by biogenic amine (BA) systems. In recent years, we have advanced our understanding on the specific neural BA pathways and receptors involved in olfactory learning and memory. However, little information exists on the contribution of cholinergic receptors to this process. Here we evaluate for the first time the proposition that, as in mammals, muscarinic ACh receptors (mAChRs) contribute to memory formation in Drosophila. Our results show that pharmacological and genetic blockade of mAChRs in MB disrupts olfactory aversive memory in larvae. This effect is not explained by an alteration in the ability of animals to respond to odorants or to execute motor programs. These results show that mAChRs in MB contribute to generating olfactory memories in Drosophila. PMID:26380118

  13. Nicotinic ACh Receptors as Therapeutic Targets in CNS Disorders

    PubMed Central

    Dineley, Kelly T.; Pandya, Anshul A.; Yakel, Jerrel L.

    2015-01-01

    The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor channels (nAChRs). These receptors are widely distributed throughout the central nervous system, being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in the mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer’s disease), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. PMID:25639674

  14. Nicotinic ACh receptors as therapeutic targets in CNS disorders.

    PubMed

    Dineley, Kelly T; Pandya, Anshul A; Yakel, Jerrel L

    2015-02-01

    The neurotransmitter acetylcholine (ACh) can regulate neuronal excitability by acting on the cys-loop cation-conducting ligand-gated nicotinic ACh receptor (nAChR) channels. These receptors are widely distributed throughout the central nervous system (CNS), being expressed on neurons and non-neuronal cells, where they participate in a variety of physiological responses such as anxiety, the central processing of pain, food intake, nicotine seeking behavior, and cognitive functions. In the mammalian brain, nine different subunits have been found thus far, which assemble into pentameric complexes with much subunit diversity; however, the α7 and α4β2 subtypes predominate in the CNS. Neuronal nAChR dysfunction is involved in the pathophysiology of many neurological disorders. Here we will briefly discuss the functional makeup and expression of the nAChRs in mammalian brain, and their role as targets in neurodegenerative diseases (in particular Alzheimer's disease, AD), neurodevelopmental disorders (in particular autism and schizophrenia), and neuropathic pain. PMID:25639674

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

    SciTech Connect

    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.

  16. Acetylcholine receptors enable the transport of rapsyn from the Golgi complex to the plasma membrane

    PubMed Central

    Park, Jee-Young; Ikeda, Hiromi; Ikenaga, Takanori; Ono, Fumihito

    2012-01-01

    The accumulation of acetylcholine receptors (AChRs) at nerve terminals is critical for signal transmission at the neuromuscular junction, and rapsyn is essential for this process. Previous studies suggest that AChRs might direct rapsyn self-clusters to the synapse. In vivo experiments with fluorescently tagged AChR or rapsyn in zebrafish larvae revealed that rapsyn self-clusters separate from AChRs did not exist before synapse formation. Examination of rapsyn in the AChR-less mutant sofa potato revealed that rapsyn in the absence of AChR was localized in the Golgi complex. Expression of muscle-type AChR in sofa potato restored synaptic clustering of rapsyn, while neuronal type AChR had no effect. To determine if this requirement of protein interaction is reciprocal, we examined the mutant twitch once, which has a missense mutation in rapsyn. While the AChRs distributed non-synaptically on the plasma membrane in twitch once, mutant rapsyn was retained in the Golgi complex. We conclude that AChRs enable the transport of rapsyn from the Golgi complex to the plasma membrane through a molecule-specific interaction. PMID:22623681

  17. Cholinergic receptor pathways involved in apoptosis, cell proliferation and neuronal differentiation

    PubMed Central

    Resende, Rodrigo R; Adhikari, Avishek

    2009-01-01

    Acetylcholine (ACh) has been shown to modulate neuronal differentiation during early development. Both muscarinic and nicotinic acetylcholine receptors (AChRs) regulate a wide variety of physiological responses, including apoptosis, cellular proliferation and neuronal differentiation. However, the intracellular mechanisms underlying these effects of AChR signaling are not fully understood. It is known that activation of AChRs increase cellular proliferation and neurogenesis and that regulation of intracellular calcium through AChRs may underlie the many functions of ACh. Intriguingly, activation of diverse signaling molecules such as Ras-mitogen-activated protein kinase, phosphatidylinositol 3-kinase-Akt, protein kinase C and c-Src is modulated by AChRs. Here we discuss the roles of ACh in neuronal differentiation, cell proliferation and apoptosis. We also discuss the pathways involved in these processes, as well as the effects of novel endogenous AChRs agonists and strategies to enhance neuronal-differentiation of stem and neural progenitor cells. Further understanding of the intracellular mechanisms underlying AChR signaling may provide insights for novel therapeutic strategies, as abnormal AChR activity is present in many diseases. PMID:19712465

  18. Mutations of Cytosolic Loop Residues Impair Assembly and Maturation of α7 Nicotinic Acetylcholine Receptors

    PubMed Central

    Mukherjee, Jayanta; Kuryatov, Alexander; Moss, Stephen J.; Lindstrom, Jon M.; Anand, Rene

    2009-01-01

    Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH-SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein RIC-3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH-SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes. PMID:19627445

  19. Analysis and modulation of the immune response of mice to acetylcholine receptor by anti-idiotypes.

    PubMed

    Souroujon, M C; Barchan, D; Fuchs, S

    1985-01-01

    Anti-idiotypes were raised in mice against three well-characterized anti-acetylcholine receptor (AChR) monoclonal antibodies (mcAbs), as well as against polyclonal mouse anti-AChR antibodies. In binding experiments, the anti-idiotypic antibodies inhibited the binding of AChR only to the immunizing idiotype. However, a less restricted specificity was found in in vivo experiments. Mice producing anti-idiotypes were challenged with AChR and the idiotypic composition of their anti-AChR response was analysed using specific rabbit anti-idiotypic antibodies. It was found that preimmunization with a certain idiotype leads to the preferential suppression of this particular idiotype in the polyclonal response to AChR. However, preimmunization with either polyclonal or monoclonal anti-AChR antibodies resulted in a reduction of the overall anti-Torpedo AChR and anti-muscle AChR titers. This reduction was greater than would be expected from the representation of each of the respective idiotypes in the polyclonal anti-AChR serum, and may imply that in addition to the immunizing idiotype other anti-AChR idiotypes are also suppressed. Our results suggest that anti-idiotypes may have a potential for the modulation of the autoimmune response directed against AChR in myasthenia. PMID:3874156

  20. Rapsyn Mutations in Humans Cause Endplate Acetylcholine-Receptor Deficiency and Myasthenic Syndrome

    PubMed Central

    Ohno, Kinji; Engel, Andrew G.; Shen, Xin-Ming; Selcen, Duygu; Brengman, Joan; Harper, C. Michel; Tsujino, Akira; Milone, Margherita

    2002-01-01

    Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with β-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn. PMID:11791205

  1. Escobar Syndrome Is a Prenatal Myasthenia Caused by Disruption of the Acetylcholine Receptor Fetal γ Subunit

    PubMed Central

    Hoffmann, Katrin; Müller, Juliane S.; Stricker, Sigmar; Megarbane, Andre; Rajab, Anna; Lindner, Tom H.; Cohen, Monika; Chouery, Eliane; Adaimy, Lynn; Ghanem, Ismat; Delague, Valerie; Boltshauser, Eugen; Talim, Beril; Horvath, Rita; Robinson, Peter N.; Lochmüller, Hanns; Hübner, Christoph; Mundlos, Stefan

    2006-01-01

    Escobar syndrome is a form of arthrogryposis multiplex congenita and features joint contractures, pterygia, and respiratory distress. Similar findings occur in newborns exposed to nicotinergic acetylcholine receptor (AChR) antibodies from myasthenic mothers. We performed linkage studies in families with Escobar syndrome and identified eight mutations within the γ-subunit gene (CHRNG) of the AChR. Our functional studies show that γ-subunit mutations prevent the correct localization of the fetal AChR in human embryonic kidney–cell membranes and that the expression pattern in prenatal mice corresponds to the human clinical phenotype. AChRs have five subunits. Two α, one β, and one δ subunit are always present. By switching γ to ɛ subunits in late fetal development, fetal AChRs are gradually replaced by adult AChRs. Fetal and adult AChRs are essential for neuromuscular signal transduction. In addition, the fetal AChRs seem to be the guide for the primary encounter of axon and muscle. Because of this important function in organogenesis, human mutations in the γ subunit were thought to be lethal, as they are in γ-knockout mice. In contrast, many mutations in other subunits have been found to be viable but cause postnatally persisting or beginning myasthenic syndromes. We conclude that Escobar syndrome is an inherited fetal myasthenic disease that also affects neuromuscular organogenesis. Because γ expression is restricted to early development, patients have no myasthenic symptoms later in life. This is the major difference from mutations in the other AChR subunits and the striking parallel to the symptoms found in neonates with arthrogryposis when maternal AChR auto-antibodies crossed the placenta and caused the transient inactivation of the AChR pathway. PMID:16826520

  2. Rapid synthesis of acetylcholine receptors at neuromuscular junctions. (Reannouncement with new availability information)

    SciTech Connect

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

    1988-12-31

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

  3. R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5

    PubMed Central

    Nakashima, Hiroaki; Ohkawara, Bisei; Ishigaki, Shinsuke; Fukudome, Takayasu; Ito, Kenyu; Tsushima, Mikito; Konishi, Hiroyuki; Okuno, Tatsuya; Yoshimura, Toshiro; Ito, Mikako; Masuda, Akio; Sobue, Gen; Kiyama, Hiroshi; Ishiguro, Naoki; Ohno, Kinji

    2016-01-01

    At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ. PMID:27328992

  4. R-spondin 2 promotes acetylcholine receptor clustering at the neuromuscular junction via Lgr5.

    PubMed

    Nakashima, Hiroaki; Ohkawara, Bisei; Ishigaki, Shinsuke; Fukudome, Takayasu; Ito, Kenyu; Tsushima, Mikito; Konishi, Hiroyuki; Okuno, Tatsuya; Yoshimura, Toshiro; Ito, Mikako; Masuda, Akio; Sobue, Gen; Kiyama, Hiroshi; Ishiguro, Naoki; Ohno, Kinji

    2016-01-01

    At the neuromuscular junction (NMJ), acetylcholine receptor (AChR) clustering is mediated by spinal motor neuron (SMN)-derived agrin and its receptors on the muscle, the low-density lipoprotein receptor-related protein 4 (LRP4) and muscle-specific receptor tyrosine kinase (MuSK). Additionally, AChR clustering is mediated by the components of the Wnt pathway. Laser capture microdissection of SMNs revealed that a secreted activator of Wnt signaling, R-spondin 2 (Rspo2), is highly expressed in SMNs. We found that Rspo2 is enriched at the NMJ, and that Rspo2 induces MuSK phosphorylation and AChR clustering. Rspo2 requires Wnt ligands, but not agrin, for promoting AChR clustering in cultured myotubes. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5), an Rspo2 receptor, is also accumulated at the NMJ, and is associated with MuSK via LRP4. Lgr5 is required for Rspo2-mediated AChR clustering in myotubes. In Rspo2-knockout mice, the number and density of AChRs at the NMJ are reduced. The Rspo2-knockout diaphragm has an altered ultrastructure with widened synaptic clefts and sparse synaptic vesicles. Frequency of miniature endplate currents is markedly reduced in Rspo2-knockout mice. To conclude, we demonstrate that Rspo2 and its receptor Lgr5 are Wnt-dependent and agrin-independent regulators of AChR clustering at the NMJ. PMID:27328992

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

    SciTech Connect

    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}.

  6. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity. PMID:26898898

  7. Expression of nicotinic acetylcholine receptor subunits in HEp-2 cells for immunodetection of autoantibody specificities in sera from Myasthenia gravis patients.

    PubMed

    George, S; Noack, M; Vanek, M; Rentzsch, J; Röber, N; Conrad, K; Roggenbuck, D; Küpper, J-H

    2015-01-01

    Myasthenia gravis (MG) is an autoimmune disease characterized by the formation of pathogenic autoantibodies mostly targeting the nicotinic acetylcholine receptor (AChR). The AChR is composed of two alpha subunits and one subunit of each beta, delta and gamma (fetal AChR), or epsilon (adult AChR), respectively. Serological diagnostics is commonly done by radioimmunoassay (RIA). Here we used an indirect immunofluorescence assay with MG patient sera on transiently transfected HEp-2 cells expressing selected components of the AChR. Our data show that already 12 out of 13 MG patient sera showed autoantibody binding to HEp-2 cells transfected to express the alpha subunit solely. Interestingly, 11 out of 13 patient sera reacted positive with cells transfected to reconstitute the complete fetal AChR, but only 6 out of 13 sera showed positive signals with cells expressing the components of adult AChR. Moreover, there was no strict correlation of the serum concentration required to obtain clear-cut fluorescence signals to the antibody titer as measured by RIA. It will be an interesting topic to further investigate if the optimal serum dilution for indirect immunofluorescence as well as the autoantibody binding preferences to defined AChR subunits and to the adult versus the fetal receptor variant could provide additional predictive value in MG diagnostics. PMID:26410878

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

    PubMed

    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

  9. Identification, characterization, and regulation of a nicotinic acetylcholine receptor on bovine adrenal chromaffin cells in culture

    SciTech Connect

    Higgins, L.S.

    1988-01-01

    Synaptic input to bovine adrenal chromaffin cells is mediated by nicotinic acetylcholine receptors (AChRs) and results in secretion of catecholamines. Three probes previously shown to recognize AChRs on neurons were used to identify the AChR on bovine adrenal chromaffin cells in culture: monoclonal antibody mAb 35, a toxin that blocks receptor function, and the agonist nicotine. Competition for {sup 3}H-nicotine binding was used to measure the affinity of cholinergic ligands, and revealed the pharmacological profile expected for a neuronal-type AChR. At steady state the rate both of receptor insertion into and loss from the plasma membrane is about 3%/hour, resulting in a half-life in the surface of about 24 hours. Exposure to the anti-AChR antibody results in a loss of AChRs from the surface of the cells through a process that has the characteristics of antigenic modulation. The number of AChRs on the surface of the chromaffin cells can also be modulated by agonists and hormones, including glucocotricoids. Catecholamines, three peptides that may be secreted by chromaffin cells, and K{sup +}-induced secretion reduce agonist-induced catecholamine release by decreasing the number of AChRs, providing a mechanism for autoregulation.

  10. Guidelines for pre-clinical assessment of the acetylcholine receptor--specific passive transfer myasthenia gravis model-Recommendations for methods and experimental designs.

    PubMed

    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

  11. CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

    PubMed Central

    Basu, Sreya; Sladecek, Stefan; Martinez de la Peña y Valenzuela, Isabel; Akaaboune, Mohammed; Smal, Ihor; Martin, Katrin; Galjart, Niels; Brenner, Hans Rudolf

    2015-01-01

    A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end–tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane. PMID:25589673

  12. A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans.

    PubMed

    Gally, Christelle; Eimer, Stefan; Richmond, Janet E; Bessereau, Jean-Louis

    2004-09-30

    Clustering neurotransmitter receptors at the synapse is crucial for efficient neurotransmission. Here we identify a Caenorhabditis elegans locus, lev-10, required for postsynaptic aggregation of ionotropic acetylcholine receptors (AChRs). lev-10 mutants were identified on the basis of weak resistance to the anthelminthic drug levamisole, a nematode-specific cholinergic agonist that activates AChRs present at neuromuscular junctions (NMJs) resulting in muscle hypercontraction and death at high concentrations. In lev-10 mutants, the density of levamisole-sensitive AChRs at NMJs is markedly reduced, yet the number of functional AChRs present at the muscle cell surface remains unchanged. LEV-10 is a transmembrane protein localized to cholinergic NMJs and required in body-wall muscles for AChR clustering. We also show that the LEV-10 extracellular region, containing five predicted CUB domains and one LDLa domain, is sufficient to rescue AChR aggregation in lev-10 mutants. This suggests a mechanism for AChR clustering that relies on extracellular protein-protein interactions. Such a mechanism is likely to be evolutionarily conserved because CUB/LDL transmembrane proteins similar to LEV-10, but lacking any assigned function, are expressed in the mammalian nervous system and might be used to cluster ionotropic receptors in vertebrates. PMID:15457263

  13. Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling.

    PubMed

    Bi, Xueyuan; He, Xi; Xu, Man; Zhao, Ming; Yu, Xiaojiang; Lu, Xingzhu; Zang, Weijin

    2015-08-01

    Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury. PMID:26066647

  14. Acetylcholine ameliorates endoplasmic reticulum stress in endothelial cells after hypoxia/reoxygenation via M3 AChR-AMPK signaling

    PubMed Central

    Bi, Xueyuan; He, Xi; Xu, Man; Zhao, Ming; Yu, Xiaojiang; Lu, Xingzhu; Zang, Weijin

    2015-01-01

    Endoplasmic reticulum (ER) stress is associated with various cardiovascular diseases. However, its pathophysiological relevance and the underlying mechanisms in the context of hypoxia/reoxygenation (H/R) in endothelial cells are not fully understood. Previous findings have suggested that acetylcholine (ACh), the major vagal nerve neurotransmitter, protected against cardiomyocyte injury by activating AMP-activated protein kinase (AMPK). This study investigated the role of ER stress in endothelial cells during H/R and explored the beneficial effects of ACh. Our results showed that H/R triggered ER stress and apoptosis in endothelial cells, evidenced by the elevation of glucose-regulated protein 78, cleaved caspase-12 and C/EBP homologous protein expression. ACh significantly decreased ER stress and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling positive cells and restored ER ultrastructural changes induced by H/R, possibly via protein kinase-like ER kinase and inositol-requiring kinase 1 pathways. Additionally, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3 AChR) inhibitor, abolished ACh-mediated increase in AMPK phosphorylation during H/R. Furthermore, M3 AChR or AMPK siRNA abrogated the ACh-elicited the attenuation of ER stress in endothelial cells, indicating that the salutary effects of ACh were likely mediated by M3 AChR-AMPK signaling. Overall, ACh activated AMPK through M3 AChR, thereby inhibited H/R-induced ER stress and apoptosis in endothelial cells. We have suggested for the first time that AMPK may function as an essential intermediate step between M3 AChR stimulation and inhibition of ER stress-associated apoptotic pathway during H/R, which may help to develop novel therapeutic approaches targeting ER stress to prevent or alleviate ischemia/reperfusion injury. PMID:26066647

  15. N-glycosylation sites on the nicotinic ACh receptor subunits regulate receptor channel desensitization and conductance.

    PubMed

    Nishizaki, Tomoyuki

    2003-06-10

    The present study investigated the effects of N-glycosylation sites on Torpedo acetylcholine (ACh) receptors expressed in Xenopus oocytes by monitoring whole-cell membrane currents and single-channel currents from excised patches. Receptors with the mutant subunit at the asparagine residue on the conserved N-glycosylation site (mbetaN141D, mgammaN141D, or mdeltaN143D) or the serine/threonine residue (mbetaT143A, mgammaS143A, or mdeltaS145A) delayed the rate of current decay as compared with wild-type receptors, and the most striking effect was found with receptors with mbetaT143A or mgammaS143A. For wild-type receptors, the lectin concanavalin A, that binds to glycosylated membrane proteins with high affinity, mimicked this effect. Receptors with mbetaN141D or mdeltaN143D exhibited lower single-channel conductance, but those with mbetaT143A, mgammaS143A, or mdeltaS145A otherwise revealed higher conductance than wild-type receptors. Mean opening time of single-channel currents was little affected by the mutation. N-glycosylation sites, thus, appear to play a role in the regulation of ACh receptor desensitization and ion permeability. PMID:12829329

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

    SciTech Connect

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

    1989-02-21

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

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

    PubMed

    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

  18. Photolabeling a Nicotinic Acetylcholine Receptor (nAChR) with an (α4)3(β2)2 nAChR-Selective Positive Allosteric Modulator.

    PubMed

    Hamouda, Ayman K; Deba, Farah; Wang, Ze-Jun; Cohen, Jonathan B

    2016-05-01

    Positive allosteric modulators (PAMs) of nicotinic acetylcholine (ACh) receptors (nAChRs) have potential clinical applications in the treatment of nicotine dependence and many neuropsychiatric conditions associated with decreased brain cholinergic activity, and 3-(2-chlorophenyl)-5-(5-methyl-1-(piperidin-4-yl)-1H-pyrrazol-4-yl)isoxazole (CMPI) has been identified as a PAM selective for neuronal nAChRs containing theα4 subunit. In this report, we compare CMPI interactions with low-sensitivity (α4)3(β2)2 and high-sensitivity (α4)2(β2)3 nAChRs, and with muscle-type nAChRs. In addition, we use the intrinsic reactivity of [(3)H]CMPI upon photolysis at 312 nm to identify its binding sites inTorpedonAChRs. Recording fromXenopusoocytes, we found that CMPI potentiated maximally the responses of (α4)3(β2)2nAChR to 10μM ACh (EC10) by 400% and with anEC50of ∼1µM. CMPI produced a left shift of the ACh concentration-response curve without altering ACh efficacy. In contrast, CMPI inhibited (∼35% at 10µM) ACh responses of (α4)2(β2)3nAChRs and fully inhibited human muscle andTorpedonAChRs with IC50values of ∼0.5µM. Upon irradiation at 312 nm, [(3)H]CMPI photoincorporated into eachTorpedo[(α1)2β1γδ] nAChR subunit. Sequencing of peptide fragments isolated from [(3)H]CMPI-photolabeled nAChR subunits established photolabeling of amino acids contributing to the ACh binding sites (αTyr(190),αTyr(198),γTrp(55),γTyr(111),γTyr(117),δTrp(57)) that was fully inhibitable by agonist and lower-efficiency, state-dependent [(3)H]CMPI photolabeling within the ion channel. Our results establish that CMPI is a potent potentiator of nAChRs containing anα4:α4 subunit interface, and that its intrinsic photoreactivy makes it of potential use to identify its binding sites in the (α4)3(β2)2nAChR. PMID:26976945

  19. Acetylcholine receptor alpha-subunit and myogenin mRNAs in thymus and thymomas.

    PubMed Central

    Kornstein, M. J.; Asher, O.; Fuchs, S.

    1995-01-01

    Myasthenia gravis is an autoimmune disorder characterized in most cases by serological antibody against the acetylcholine receptor (AChR). Evidence for intrathymic localization of AChR suggests that the thymus has an important role in the pathogenesis of this disorder. Using reverse transcription followed by the polymerase chain reaction, we have demonstrated AChR alpha-subunit mRNA in thymuses and thymomas from patients with and without myasthenia gravis. We have also studied the expression of myogenin which is known to be involved in the regulation of AChR expression. By using the reverse transcription polymerase chain reaction, we found myogenin mRNAs in all of the thymuses and thymomas. Thus, both AChR alpha-subunit and myogenin mRNA are present in all of these specimens. By immunohistochemistry myoid cells (desmin and myoglobin positive) were present in all (four of four) thymuses studied and in two of five thymomas. Thus, in thymomas, nonmyoid cells might express both AChR and myogenin. These results indicate that cells within the thymus and thymoma express AChR and its regulatory protein myogenin and that such cells, under certain conditions, might play a role in the triggering of myasthenia gravis. Images Figure 2 Figure 3 PMID:7778671

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

    PubMed

    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

  1. Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor.

    PubMed

    Barchan, D; Asher, O; Tzartos, S J; Fuchs, S; Souroujon, M C

    1998-02-01

    Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR alpha subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR alpha subunit (H alpha1-210), and to portions of it that encompass either the MIR (H alpha1-121) or the ligand binding site of AChR (H alpha122-210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments H alpha1-121 and H alpha1-210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments H alpha1-121 and H alpha1-210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment H alpha122-210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia. PMID:9521072

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

    SciTech Connect

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

    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.

  3. The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

    PubMed

    Barchan, D; Ovadia, M; Kochva, E; Fuchs, S

    1995-07-18

    The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7619817

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

    PubMed

    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

  5. Standardization of the experimental autoimmune myasthenia gravis (EAMG) model by immunization of rats with Torpedo californica acetylcholine receptors — Recommendations for methods and experimental designs

    PubMed Central

    Losen, Mario; Martinez-Martinez, Pilar; Molenaar, Peter C.; Lazaridis, Konstantinos; Tzartos, Socrates; Brenner, Talma; Duan, Rui-Sheng; Luo, Jie; Lindstrom, Jon; Kusner, Linda

    2015-01-01

    Myasthenia gravis (MG) with antibodies against the acetylcholine receptor (AChR) is characterized by a chronic, fatigable weakness of voluntary muscles. The production of autoantibodies involves the dysregulation of T cells which provide the environment for the development of autoreactive B cells. The symptoms are caused by destruction of the postsynaptic membrane and degradation of the AChR by IgG autoantibodies, predominantly of the G1 and G3 subclasses. Active immunization of animals with AChR from mammalian muscles, AChR from Torpedo or Electrophorus electric organs, and recombinant or synthetic AChR fragments generates a chronic model of MG, termed experimental autoimmune myasthenia gravis (EAMG). This model covers cellular mechanisms involved in the immune response against the AChR, e.g. antigen presentation, T cell-help and regulation, B cell selection and differentiation into plasma cells. Our aim is to define standard operation procedures and recommendations for the rat EAMG model using purified AChR from the Torpedo californica electric organ, in order to facilitate more rapid translation of preclinical proof of concept or efficacy studies into clinical trials and, ultimately, clinical practice. PMID:25796590

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

    PubMed

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

    2014-07-25

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

  7. Wnt proteins regulate acetylcholine receptor clustering in muscle cells

    PubMed Central

    2012-01-01

    Background The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. Results In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. Conclusions These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4. PMID:22309736

  8. Agonists with supraphysiological efficacy at the muscarinic M2 ACh receptor

    PubMed Central

    Schrage, R; Seemann, WK; Klöckner, J; Dallanoce, C; Racké, K; Kostenis, E; De Amici, M; Holzgrabe, U; Mohr, K

    2013-01-01

    Background and Purpose Artificial agonists may have higher efficacy for receptor activation than the physiological agonist. Until now, such ‘superagonism’ has rarely been reported for GPCRs. Iperoxo is an extremely potent muscarinic receptor agonist. We hypothesized that iperoxo is a ‘superagonist’. Experimental Approach Signalling of iperoxo and newly synthesized structural analogues was compared with that of ACh at label-free M2 muscarinic receptors applying whole cell dynamic mass redistribution, measurement of G-protein activation, evaluation of cell surface agonist binding and computation of operational efficacies. Key Results In CHO-hM2 cells, iperoxo significantly exceeds ACh in Gi/Gs signalling competence. In the orthosteric loss-of-function mutant M2-Y1043.33A, the maximum effect of iperoxo is hardly compromised in contrast to ACh. ‘Superagonism’ is preserved in the physiological cellular context of MRC-5 human lung fibroblasts. Structure–signalling relationships including iperoxo derivatives with either modified positively charged head group or altered tail suggest that ‘superagonism’ of iperoxo is mechanistically based on parallel activation of the receptor protein via two orthosteric interaction points. Conclusion and Implications Supraphysiological agonist efficacy at muscarinic M2 ACh receptors is demonstrated for the first time. In addition, a possible underlying molecular mechanism of GPCR ‘superagonism’ is provided. We suggest that iperoxo-like orthosteric GPCR activation is a new avenue towards a novel class of receptor activators. Linked Article This article is commented on by Langmead and Christopoulos, pp. 353–356 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12142 PMID:23062057

  9. The fetal form of the acetylcholine receptor distinguishes rhabdomyosarcomas from other childhood tumors.

    PubMed Central

    Gattenloehner, S.; Vincent, A.; Leuschner, I.; Tzartos, S.; Müller-Hermelink, H. K.; Kirchner, T.; Marx, A.

    1998-01-01

    The fetal nicotinic acetylcholine receptor (AChR) of muscle is an oligomeric membrane protein with subunit composition alpha2betadeltagamma. After birth, the adult form, in which an epsilon-subunit replaces the gamma-subunit, predominates, and expression of the fetal form is limited to thymic myoid cells, extraocular muscles, and denervated striated muscle. We looked for expression of AChR in rhabdomyosarcomas and other childhood tumors by reverse transcription polymerase chain reaction and immunohistochemistry. mRNA for the AChR gamma-subunit was detected in all embryonal and alveolar rhabdomyosarcomas tested (n = 16) and in some tumors with a rhabdomyomatous component (n = 2) but not in other nonrhabdomyomatous tumors of childhood and adults (n = 45). The fetal form of the AChR was detected immunohistochemically in five of eight embryonal and four of eight alveolar rhabdomyosarcomas and in two Wilms' tumors with a rhabdomyomatous component but not in other tumors or in normal muscle. We conclude that reverse transcription polymerase chain reaction for AChR gamma-subunit could be useful for the diagnosis of rhabdomyosarcoma of childhood and for the detection of micrometastases and minimal residual disease. In addition, the fetal AChR protein is the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle. Our findings, therefore, imply that the fetal AChR may be a target for in vivo imaging and, as AChR internalization and degradation is increased by antibody-induced cross-linking, may also provide a sensitive and specific target for immunotherapeutic strategies. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:9466570

  10. Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.

    PubMed

    Wakkach, A; Guyon, T; Bruand, C; Tzartos, S; Cohen-Kaminsky, S; Berrih-Aknin, S

    1996-10-15

    The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG. PMID:8871679

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

    PubMed

    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

  12. Identification of epitopes within a highly immunogenic region of acetylcholine receptor by a phage epitope library.

    PubMed

    Barchan, D; Balass, M; Souroujon, M C; Katchalski-Katzir, E; Fuchs, S

    1995-11-01

    We have employed a hexapeptide phage-epitope library to identify epitopes for a mAb (mAb 5.14), which is directed to a determinant within a highly immunogenic, cytoplasmic region of the alpha-subunit of acetylcholine receptor (AChR). We have selected two different peptide-presenting phages (SWDDIR-phage and LWILTR-phage) which interact specifically with mAb 5.14. This interaction is specifically inhibited by AChR and by synthetic peptides corresponding to the hexapeptides presented by the selected phages. Although mAb 5.14 binds to AChR in its native as well as its denatured form, the selected hexapeptides do not exist as such in the AChR molecule. However, three amino acid sequence homologies with these hexapeptides were shown to be present in the cytoplasmic region of Torpedo AChR. By extending the selected hexapeptides, at one or both ends, with amino acid residues flanking the hexapeptides in the phage, we obtained mimotopes with an up to two order of magnitude higher affinity to the Ab. These extended peptides were able to efficiently block the binding of mAb 5.14 to both peptide-presenting phages, and to AChR. PMID:7594584

  13. The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

    PubMed Central

    Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

    2014-01-01

    BACKGROUND AND PURPOSE Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

  14. The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain

    PubMed Central

    Mazhar, Sania; Herbst, Ruth

    2012-01-01

    Efficient synaptic transmission at the neuromuscular junction (NMJ) requires the topological maturation of the postsynaptic apparatus from an oval acetylcholine receptor (AChR)-rich plaque into a complex pretzel-shaped array of branches. However, compared to NMJ formation very little is known about the mechanisms that regulate NMJ maturation. Recently the process of in vivo transformation from plaque into pretzel has been reproduced in vitro by culturing myotubes aneurally on laminin-coated substrate. It was proposed that the formation of complex AChR clusters is regulated by a MuSK-dependent muscle intrinsic program. To elucidate the structure–function role of MuSK in the aneural maturation of AChR pretzels, we used muscle cell lines expressing MuSK mutant and chimeric proteins. Here we report, that besides its role during agrin-induced AChR clustering, MuSK kinase activity is also necessary for substrate-dependent cluster formation. Constitutive-active MuSK induces larger AChR clusters, a faster cluster maturation on laminin and increases the anchorage of AChRs to the cytoskeleton compared to MuSK wild-type. In addition, we find that the juxtamembrane region of MuSK, which has previously been shown to regulate agrin-induced AChR clustering, is unable to induce complex AChR clusters on laminin substrate. Most interestingly, MuSK kinase activity is not sufficient for laminin-dependent AChR cluster formation since the MuSK ectodomain is also required suggesting a so far undiscovered instructive role for the extracellular domain of MuSK. PMID:22210232

  15. The role of the amino acid residue at α1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors

    PubMed Central

    Purohit, P G; Tate, R J; Pow, E; Hill, D; Connolly, J G

    2007-01-01

    Background and Purpose: Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. Experimental Approach: A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [125I]α-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant α1 subunits. Key results: The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC50 results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC50 values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at α1:189 and proline at α1:194 to serine. However the α1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. Conclusions and implications: The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The α1:Phe189Ser substitution, but not α1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC50 values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary ‘reinforcement'. PMID:17293883

  16. Neural regulation of MRNA for the alpha-subunit of acetylcholine receptors: Role of neuromuscular transmission. (Reannouncement with new availability information)

    SciTech Connect

    Lipsky, N.G.; Drachman, D.B.; Pestronk, A.; Shih, P.J.

    1989-12-31

    Levels of mRNA for acetylcholine receptor (AChR) subunits are relatively low in innervated skeletal muscles. Following denervation they rise rapidly, leading to increased AChR synthesis. The mechanism by which motor nerves normally regulate these mRNA levels is not yet known. In order to determine the possible role of synaptic transmission in this process, the authors have compared the effect of blockade of cholinergic ACh transmission with that of surgical denervation. Blockade of quantal ACh transmission was produced by injection of type A botulinum toxin into the soleus muscles of rats.

  17. Voltage-dependent interaction between the muscarinic ACh receptor and proteins of the exocytic machinery.

    PubMed Central

    Linial, M; Ilouz, N; Parnas, H

    1997-01-01

    1. Release of neurotransmitter into the synaptic cleft is the last step in the chain of molecular events following the arrival of an action potential at the nerve terminal. The neurotransmitter exerts negative feedback on its own release. This inhibition would be most effective if exerted on the first step in this chain of events, i.e. a step that is mediated by membrane depolarization. Indeed, in numerous studies feedback inhibition was found to be voltage dependent. 2. The purpose of this study is to investigate whether the mechanism underlying feedback inhibition of transmitter release resides in interaction between the presynaptic autoreceptors and the exocytic apparatus, specifically the soluble NSF-attachment protein receptor (SNARE) complex. 3. Using rat synaptosomes we show that the muscarinic ACh autoreceptor (mAChR) is an integral component of the exocytic machinery. It interacts with syntaxin, synaptosomal-associated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein (VAMP) and synaptotagmin as shown using both cross-linking and immunoprecipitation. 4. The interaction between mAChRs and both syntaxin and SNAP-25 is modulated by depolarization levels; binding is maximal at resting potential and disassembly occurs at higher depolarization. 5. This voltage-dependent interaction of mAChRs with the secretory core complex appears suitable for controlling the rapid, synchronous neurotransmitter release at nerve terminals. Images Figure 2 Figure 3 PMID:9365901

  18. Transient Cholesterol Effects on Nicotinic Acetylcholine Receptor Cell-Surface Mobility

    PubMed Central

    Almarza, Gonzalo; Sánchez, Francisco; Barrantes, Francisco J.

    2014-01-01

    To what extent do cholesterol-rich lipid platforms modulate the supramolecular organization of the nicotinic acetylcholine receptor (AChR)? To address this question, the dynamics of AChR particles at high density and its cholesterol dependence at the surface of mammalian cells were studied by combining total internal reflection fluorescence microscopy and single-particle tracking. AChR particles tagged with a monovalent ligand, fluorescent α-bungarotoxin (αBTX), exhibited two mobile pools: i) a highly mobile one undergoing simple Brownian motion (16%) and ii) one with restricted motion (∼50%), the rest being relatively immobile (∼44%). Depletion of membrane cholesterol by methyl-α-cyclodextrin increased the fraction of the first pool to 22% and 33% after 15 and 40 min, respectively; the pool undergoing restricted motion diminished from 50% to 44% and 37%, respectively. Monoclonal antibody binding results in AChR crosslinking-internalization after 2 h; here, antibody binding immobilized within minutes ∼20% of the totally mobile AChR. This proportion dramatically increased upon cholesterol depletion, especially during the initial 10 min (83.3%). Thus, antibody crosslinking and cholesterol depletion exhibited a mutually synergistic effect, increasing the average lifetime of cell-surface AChRs∼10 s to ∼20 s. The instantaneous (microscopic) diffusion coefficient D2–4 of the AChR obtained from the MSD analysis diminished from ∼0.001 µm2 s−1 to ∼0.0001–0.00033 µm2 s−1 upon cholesterol depletion, ∼30% of all particles falling into the stationary mode. Thus, muscle-type AChR exhibits heterogeneous motional regimes at the cell surface, modulated by the combination of intrinsic (its supramolecular organization) and extrinsic (membrane cholesterol content) factors. PMID:24971757

  19. Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

    PubMed

    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

  20. A role for the CaM Kinase II related anchoring protein (αkap) in maintaining the stability of nicotinic Acetylcholine Receptors

    PubMed Central

    Mouslim, Chakib; Aittaleb, Mohamed; Hume, Richard I.; Akaaboune, Mohammed

    2012-01-01

    αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function. PMID:22496563

  1. Riluzole blocks human muscle acetylcholine receptors

    PubMed Central

    Deflorio, Cristina; Palma, Eleonora; Conti, Luca; Roseti, Cristina; Manteca, Alessia; Giacomelli, Elena; Catalano, Myriam; Limatola, Cristina; Inghilleri, Maurizio; Grassi, Francesca

    2012-01-01

    Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α1β1δ subunits plus the γ or ɛ subunit (γ- or ɛ-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 μm, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ɛ-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated. PMID:22431338

  2. Rectification of muscle and nerve deficits in paralyzed ryanodine receptor type 1 mutant embryos

    PubMed Central

    Hanson, M. Gartz; Niswander, Lee A.

    2015-01-01

    Locomotion and respiration require motor axon connectivity and activation of the neuromuscular junction (NMJ). Through a forward genetic screen for muscle weakness, we recently reported an allele of ryanodine receptor type 1 (Ryr1AG). Here we reveal a role for functional RyR1 during acetylcholine receptor (AChR) cluster formation and embryonic synaptic transmission. Ryr1AG homozygous embryos are non-motile. Motor axons extend past AChR clusters and enlarged AChR clusters are found under fasciculated nerves. Using physiological and pharmacological methods, we show that contractility can be resumed through the masking of a potassium leak, and evoked vesicular release can be resumed via bypassing the defect in RyR1 induced calcium release. Moreover, we show the involvement of ryanodine receptors in presynaptic release at the NMJ. This data provides evidence of a role for RyR1 on both the pre- and postsynaptic sides of the NMJ. PMID:26025922

  3. Mechanism of nasal tolerance induced by a recombinant fragment of acetylcholine receptor for treatment of experimental myasthenia gravis.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    2000-11-01

    Acetylcholine receptor (AChR) is the major autoantigen in myasthenia gravis (MG) and experimental autoimmune MG (EAMG). Here we analyze the mechanisms involved in suppression of ongoing EAMG in rats by nasal administration of a recombinant fragment from the human AChR alpha-subunit. We demonstrate that such a fragment, expressed without a fusion partner, confers nasal tolerance that can be adoptively transferred. Our observations suggest that the underlying mechanism of this nasal tolerance is active suppression involving a shift from a Th1 to a Th2/Th3-regulated AChR-specific response which may be mediated by down regulation of costimulatory factors. PMID:11063834

  4. A study of brain insulin receptors, AChE activity and oxidative stress in rat model of ICV STZ induced dementia.

    PubMed

    Agrawal, Rahul; Tyagi, Ethika; Shukla, Rakesh; Nath, Chandishwar

    2009-03-01

    In the present study, role of brain insulin receptors (IRs) in memory functions and its correlation with acetylcholinesterase (AChE) activity and oxidative stress in different brain regions were investigated in intracerebroventricular (ICV) streptozotocin (STZ) induced dementia model. Rats were treated with STZ (3 mg/kg, ICV) on day 1 and 3. Donepezil (5 mg/kg po) and melatonin (20 mg/kg ip) were administered in pre- and post-treatment schedules. Morris water maze test was done on day 14 and animals were sacrificed on day 21 from 1st STZ injection. Memory deficit was found in STZ group as indicated by no significant decrease in latency time antagonized by donepezil and melatonin. IR protein level was found significantly increased in trained group as compared to control, whereas STZ decreased IR level significantly as compared to trained rats in hippocampus which indicates that IR is associated with memory functions. STZ induced decrease in IR was reversed by melatonin but not by donepezil. Melatonin per se did not show any significant change in IR level as compared to control. AChE activity (DS and SS fraction) was found to be increased in hippocampus in STZ group as compared to trained which was inhibited by donepezil and melatonin. Increase in MDA level and decrease in GSH level were obtained in STZ group indicating oxidative stress, which was attenuated by donepezil and melatonin. Effectiveness of antioxidant, melatonin but not of anti-cholinesterase, donepezil against STZ induced changes in IR indicates that IR is more affected with oxidative stress than cholinergic changes. PMID:19705549

  5. Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells.

    PubMed Central

    Yuan, Y; Axelrod, D

    1995-01-01

    Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules on living, nondeoxygenated cells with characteristic times previously ranging from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rotating membrane protein monomers that typically should have a characteristic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With this new system, PFRAP experiments can be conducted with sample time as short as 0.4 microseconds and detection of possible characteristic times of less than 2 microseconds. The system is tested on rhodamine-alpha-bungarotoxin-labeled acetylcholine receptors (AChRs) on myotubes grown in primary cultures of embryonic rat muscle, in both endogenously clustered and nonclustered regions of AChR distribution. It is found that approximately 40% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian motion. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregating factors) on the myotube AChR were also examined by the fast PFRAP system. Brain extract is known to abolish the presence of endogenous clusters and to induce the formation of new clusters. It is found here that rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotational diffusion in the nonclustered regions of extract-treated myotubes remains rapid. Images FIGURE 3 PMID:8527682

  6. Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.

    PubMed Central

    Protti, M P; Manfredi, A A; Straub, C; Howard, J F; Conti-Tronconi, B M

    1990-01-01

    In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation. Images PMID:2145582

  7. Regulation of acetylcholine receptor alpha subunit variants in human myasthenia gravis. Quantification of steady-state levels of messenger RNA in muscle biopsy using the polymerase chain reaction.

    PubMed Central

    Guyon, T; Levasseur, P; Truffault, F; Cottin, C; Gaud, C; Berrih-Aknin, S

    1994-01-01

    Myasthenia gravis (MG) is an autoimmune disease mediated by auto-antibodies that attack the nicotinic acetylcholine receptor (AChR). To elucidate the molecular mechanisms underlying the decrease in AChR levels at the neuromuscular junction, we investigated the regulation of AChR expression by analyzing mRNA of the two AChR alpha subunit isoforms (P3A+ and P3A-) in muscle samples from myasthenic patients relative to controls. We applied a quantitative method based on reverse transcription of total RNA followed by polymerase chain reaction (PCR), using an internal standard we constructed by site-directed mutagenesis. An increased expression of mRNA coding for the alpha subunit of the AChR isoforms was observed in severely affected patients (P < 0.003 versus controls) but not in moderately affected patients, independently of the anti-AChR antibody titer. Study of mRNA precursor levels indicates a higher expression in severely affected patients compared to controls, suggesting an enhanced rate of transcription of the message coding for the alpha subunit isoforms in these patients. We have also reported that mRNA encoding both isoforms are expressed at an approximate 1:1 ratio in controls and in patients. We have thus identified a new biological parameter correlated with disease severity, and provide evidence of a compensatory mechanism to balance the loss of AChR in human myasthenia gravis, which is probably triggered only above a certain degree of AChR loss. Images PMID:8040257

  8. Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn–plectin 1f complexes

    PubMed Central

    Mihailovska, Eva; Raith, Marianne; Valencia, Rocio G.; Fischer, Irmgard; Banchaabouchi, Mumna Al; Herbst, Ruth; Wiche, Gerhard

    2014-01-01

    Mutations in the cytolinker protein plectin lead to grossly distorted morphology of neuromuscular junctions (NMJs) in patients suffering from epidermolysis bullosa simplex (EBS)-muscular dystrophy (MS) with myasthenic syndrome (MyS). Here we investigated whether plectin contributes to the structural integrity of NMJs by linking them to the postsynaptic intermediate filament (IF) network. Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells. We found plectin isoform 1f (P1f) to bridge AChRs and IFs via direct interaction with the AChR-scaffolding protein rapsyn in an isoform-specific manner; forced expression of P1f in plectin-deficient cells rescued both compromised AChR clustering and IF network anchoring. In conditional plectin knockout mice with gene disruption in muscle precursor/satellite cells (Pax7-Cre/cKO), uncoupling of AChRs from IFs was shown to lead to loss of postsynaptic membrane infoldings and disorganization of the NMJ microenvironment, including its invasion by microtubules. In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span. Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion. PMID:25318670

  9. Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail, Helix pomatia L. Tentacle

    PubMed Central

    Kiss, Tibor; Krajcs, Nóra; Pirger, Zsolt; Hernádi, László

    2014-01-01

    Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na+ and Ca2+ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery. PMID:25303328

  10. Evidence from the anti-idiotypic network that the acetylcholine receptor is a rabies virus receptor.

    PubMed Central

    Hanham, C A; Zhao, F; Tignor, G H

    1993-01-01

    We have developed idiotype-anti-idiotype monoclonal antibodies that provide evidence for rabies virus binding to the acetylcholine receptor (AChR). Hybridoma cell lines 7.12 and 7.25 resulted after fusion of NS-1 myeloma cells with spleen cells from a BALB/c mouse immunized with rabies virus strain CVS. Antibody 7.12 reacted with viral glycoprotein and neutralized virus infectivity in vivo. It also neutralized infectivity in vitro when PC12 cells, which express neuronal AChR, but not CER cells or neuroblastoma cells (clone N18), which have no AChR, were used. Antibody 7.25 reacted with nucleocapsid protein. Anti-idiotypic monoclonal antibody B9 was produced from fusion of NS-1 cells with spleen cells from a mouse immunized with 7.12 Fab. In an enzyme-linked immunosorbent assay and immunoprecipitation, B9 reacted with 7.12, polyclonal rabies virus immune dog serum, and purified AChR. The binding of B9 to 7.12 and immune dog serum was inhibited by AChR. B9 also inhibited the binding of 7.12 to rabies virus both in vitro and in vivo. Indirect immunofluorescence revealed that B9 reacted at neuromuscular junctions of mouse tissue. B9 also reacted in indirect immunofluorescence with distinct neurons in mouse and monkey brain tissue as well as with PC12 cells. B9 staining of neuronal elements in brain tissue of rabies virus-infected mice was greatly reduced. Rabies virus inhibited the binding of B9 to PC12 cells. Mice immunized with B9 developed low-titer rabies virus-neutralizing antibody. These mice were protected from lethal intramuscular rabies virus challenge. In contrast, anti-idiotypic antibody raised against nucleocapsid antibody 7.25 did not react with AChR. Images PMID:7677960

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

    PubMed

    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

  12. Effects of preganglionic denervation and postganglionic axotomy on acetylcholine receptors in the chick ciliary ganglion

    PubMed Central

    1987-01-01

    The regulation of nicotinic acetylcholine receptors (AChRs) in chick ciliary ganglia was examined by using a radiolabeled anti-AChR mAb to quantitate the amount of receptor in ganglion detergent extracts after preganglionic denervation or postganglionic axotomy. Surgical transection of the preganglionic input to the ciliary ganglion in newly hatched chicks caused a threefold reduction in the total number of AChRs within 10 d compared with that present in unoperated contralateral control ganglia. Surgical transection of both the choroid and ciliary nerves emerging from the ciliary ganglion in newly hatched chicks to establish postganglionic axotomy led to a nearly 10-fold reduction in AChRs within 5 d compared with unoperated contralateral ganglia. The declines were specific since they could not be accounted for by changes in ganglionic protein or by decreases in neuronal survival or size. Light microscopy revealed no gross morphological differences between neurons in operated and control ganglia. A second membrane component of cholinergic relevance on chick ciliary ganglion neurons is the alpha-bungarotoxin (alpha-Bgt)-binding component. The alpha-Bgt-binding component also declined in number after either postganglionic axotomy or preganglionic denervation, but appeared to do so with a more rapid time course than did ganglionic AChRs. The results imply that cell-cell interactions in vivo specifically regulate both the number of AChRs and the number of alpha-Bgt-binding components in the ganglion. Regulation of these neuronal cholinergic membrane components clearly differs from that previously described for muscle AChRs. PMID:3667699

  13. Wnt signals organize synaptic prepattern and axon guidance through the zebrafish unplugged/MuSK receptor.

    PubMed

    Jing, Lili; Lefebvre, Julie L; Gordon, Laura R; Granato, Michael

    2009-03-12

    Early during neuromuscular development, acetylcholine receptors (AChRs) accumulate at the center of muscle fibers, precisely where motor growth cones navigate and synapses eventually form. Here, we show that Wnt11r binds to the zebrafish unplugged/MuSK ectodomain to organize this central muscle zone. In the absence of such a zone, prepatterned AChRs fail to aggregate and, as visualized by live-cell imaging, growth cones stray from their central path. Using inducible unplugged/MuSK transgenes, we show that organization of the central muscle zone is dispensable for the formation of neural synapses, but essential for AChR prepattern and motor growth cone guidance. Finally, we show that blocking noncanonical dishevelled signaling in muscle fibers disrupts AChR prepatterning and growth cone guidance. We propose that Wnt ligands activate unplugged/MuSK signaling in muscle fibers to restrict growth cone guidance and AChR prepatterns to the muscle center through a mechanism reminiscent of the planar cell polarity pathway. PMID:19285469

  14. Immobilization with Atrophy Induces Increased Expression of Neuronal Nicotinic α7 Acetylcholine Receptors in Muscle Contributing to Neurotransmission

    PubMed Central

    Lee, Sangseok; Yang, Hong-seuk; Sasakawa, Tomoki; Khan, Mohammed A. S.; Khatri, Ashok; Kaneki, Masao; Jeevendra Martyn, J. A.

    2014-01-01

    Background Mature acetylcholine receptor (AChR) isoform normally mediates muscle contraction. The hypothesis that α7AChRs upregulate during immobilization and contribute to neurotransmission was tested pharmacologically using specific blockers to mature (waglerin-1), immature (αA-OIVA), and α7AChRs methyllycaconitine, and non-specific muscle AChR antagonist, α-bungarotoxin. Methods Mice were immobilized; contralateral limb was control. Fourteen days later, anesthetized mice were mechanically ventilated. Nerve-stimulated tibialis muscle contractions on both sides were recorded, and blockers enumerated above sequentially administered via jugular vein. Data are mean ± S.E. Results Immobilization (N=7) induced tibialis muscle atrophy (40.6 ± 2.8 vs 52.1 ± 2.0 mg, p<0.01) and decrease of twitch tension (34.8 ± 1.1 vs. 42.9 ± 1.5 g, p< 0.01). Waglerin-1 (0.3 ± 0.05 μg/g) significantly (p=0.001, N=9) depressed twitch tension on contralateral (≥ 97%) versus immobilized side (~45%). Additional waglerin-1 (total dose 1.06 ± 0.12 μg/g or ~15.0 X ED50 in normals) could not depress twitch ≥ 80% on immobilized side. Immature AChR blocker, αA-OIVA (17.0 ± 0.25 μg/g) did not change tension bilaterally. Administration of α-bungarotoxin (N=4) or methyllycaconitine (N=3) caused ≥ 96% suppression of the remaining twitch tension on immobilized side. Methyllycaconitine, administered first (N=3), caused equipotent inhibition by waglerin-1 on both sides. Protein expression of α7AChRs was significantly (N=3, p<0.01) increased on the immobilized side. Conclusions Ineffectiveness of waglerin-1 suggests the twitch tension during immobilization is maintained by receptors other than mature AChRs. Since αA-OIVA caused no changes, immature AChRs contribute minimally to neurotransmission. During immobilization ~20% of twitch tension is maintained by upregulation of α-bungarotoxin- and methyllycaconitine-sensitive α7AChRs. PMID:24126263

  15. Activation of endplate nicotinic acetylcholine receptors by agonists.

    PubMed

    Auerbach, Anthony

    2015-10-15

    The interaction of a small molecule made in one cell with a large receptor made in another is the signature event of cell signaling. Understanding the structure and energy changes associated with agonist activation is important for engineering drugs, receptors and synapses. The nicotinic acetylcholine receptor (AChR) is a ∼300kD ion channel that binds the neurotransmitter acetylcholine (ACh) and other cholinergic agonists to elicit electrical responses in the central and peripheral nervous systems. This mini-review is in two sections. First, general concepts of skeletal muscle AChR operation are discussed in terms of energy landscapes for conformational change. Second, adult vs. fetal AChRs are compared with regard to interaction energies between ACh and agonist-site side chains, measured by single-channel electrophysiology and molecular dynamics simulations. The five aromatic residues that form the core of each agonist binding site can be divided into two working groups, a triad (led by αY190) that behaves similarly at all sites and a coupled pair (led by γW55) that has a large influence on affinity only in fetal AChRs. Each endplate AChR has 5 homologous subunits, two of α(1) and one each of β, δ, and either γ (fetal) or ϵ (adult). These nicotinic AChRs have only 2 functional agonist binding sites located in the extracellular domain, at αδ and either αγ or αϵ subunit interfaces. The receptor undergoes a reversible, global isomerization between structures called C and O. The C shape does not conduct ions and has a relatively low affinity for ACh, whereas O conducts cations and has a higher affinity. When both agonist sites are empty (filled only with water) the probability of taking on the O conformation (PO) is low, <10(-6). When ACh molecules occupy the agonist sites the C→O opening rate constant and C↔O gating equilibrium constant increase dramatically. Following a pulse of ACh at the nerve-muscle synapse, the endplate current rises rapidly

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

    PubMed Central

    Ren, Zuo Jun; Mummalaneni, Shobha; Qian, Jie; Baumgarten, Clive M.; DeSimone, John A.; Lyall, Vijay

    2015-01-01

    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. PMID:26039516

  17. Arecoline inhibits and destabilizes agrin-induced acetylcholine receptor cluster formation in C2C12 myotubes.

    PubMed

    Chang, Yung-Fu; Liu, Ting-Yuan; Liu, Shao-Tung

    2013-10-01

    Areca nut (Areca catechu) is chewed as a medical and psychoactive food by roughly 10% of the world population. Areca nut chewing may lead to low birth weight, premature delivery and impaired muscle development. Our previous study showed that arecoline, a major alkaloid in the areca nut, inhibited the myogenic differentiation of C2C12 myoblastic cells. The clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane at the neuromuscular junction (NMJ) by agrin, a signaling protein released by motor neurons, is critical for the development of functional muscles. Here, we further investigate whether arecoline affects the AChR clustering using cultured C2C12 myotubes. Rhodamine-conjugated α-bungarotoxin was used to detect the presence of AChR clusters. Our results showed that arecoline inhibited the formation of agrin-induced AChR clusters and destabilized agrin-induced or spontaneous AChR cluster formation. In addition, arecoline inhibited the expression of myogenin in C2C12 myotubes. These results shed light on the important role of arecoline on the detrimental effect of areca nut to muscle development. PMID:23933062

  18. The ligand binding domain of the nicotinic acetylcholine receptor. Immunological analysis.

    PubMed

    Kachalsky, S G; Aladjem, M; Barchan, D; Fuchs, S

    1993-03-01

    The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with alpha-bungarotoxin (alpha-BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR alpha-subunit binds alpha-BTX, whereas the mongoose-expressed domain is not recognized by alpha-BTX. On the other hand, both the mouse and mongoose domains bind to the site-specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of alpha-BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other. PMID:8440381

  19. Vector-averaged gravity does not alter acetylcholine receptor single channel properties

    NASA Technical Reports Server (NTRS)

    Reitstetter, R.; Gruener, R.

    1994-01-01

    To examine the physiological sensitivity of membrane receptors to altered gravity, we examined the single channel properties of the acetylcholine receptor (AChR), in co-cultures of Xenopus myocytes and neurons, to vector-averaged gravity in the clinostat. This experimental paradigm produces an environment in which, from the cell's perspective, the gravitational vector is "nulled" by continuous averaging. In that respect, the clinostat simulates one aspect of space microgravity where the gravity force is greatly reduced. After clinorotation, the AChR channel mean open-time and conductance were statistically not different from control values but showed a rotation-dependent trend that suggests a process of cellular adaptation to clinorotation. These findings therefore suggest that the ACHR channel function may not be affected in the microgravity of space despite changes in the receptor's cellular organization.

  20. Effect of nicotinic acetylcholine receptor alpha 1 (nAChRα1) peptides on rabies virus infection in neuronal cells.

    PubMed

    Sajjanar, Basavaraj; Saxena, Shikha; Bisht, Deepika; Singh, Arvind Kumar; Manjunatha Reddy, G B; Singh, Rajendra; Singh, R P; Kumar, Satish

    2016-06-01

    Rabies virus (RABV) is neurotropic and causes acute progressive encephalitis. Herein, we report the interaction of nAChRα1-subunit peptides with RABV and the effect of these peptides on RABV infection in cultured neuronal cells. Peptide sequences derived from torpedo, bovine, human and rats were synthesized and studied for their interactions with RABV using virus capture ELISA and peptide immunofluorescence. The results showed specific binding of the nAChRα1-subunit peptides to the RABV. In the virus adsorption assay, these peptides were found to inhibit the attachment of the RABV to the neuronal cells. The nAChRα1-subunit peptides inhibited the RABV infection and reduced viral gene expression in the cultured neuroblastoma (N2A) cells. Torpedo peptide sequence (T-32) had highest antiviral effect (IC50=14±3.01μM) compared to the other peptides studied. The results of the study indicated that nAChRα1-subunit peptides may act as receptor decoy molecules and inhibit the binding of virus to the native host cell receptors and hence may reduce viral infection. PMID:26656837

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

    PubMed

    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

  2. Sequence and functional expression of a single alpha subunit of an insect nicotinic acetylcholine receptor.

    PubMed Central

    Marshall, J; Buckingham, S D; Shingai, R; Lunt, G G; Goosey, M W; Darlison, M G; Sattelle, D B; Barnard, E A

    1990-01-01

    We report the isolation and sequence of a cDNA clone that encodes a locust (Schistocerca gregaria) nervous system nicotinic acetylcholine receptor (AChR) subunit (alpha L1). The calculated molecular weight of the unglycosylated polypeptide, which contains in the proposed extracellular domain two adjacent cysteine residues which are characteristic of alpha (ligand binding) subunits, is 60,641 daltons. Injection into Xenopus oocytes, of RNA synthesized from this clone in vitro, results in expression of functional nicotinic receptors in the oocyte membrane. In these, nicotine opens a cation channel; the receptors are blocked by both alpha-bungarotoxin (alpha-Bgt) and kappa-bungarotoxin (kappa-Bgt). Reversible block of the expressed insect AChR by mecamylamine, d-tubocurarine, tetraethylammonium, bicuculline and strychnine has also been observed. These data are entirely consistent with previously reported electrophysiological studies on in vivo insect nicotinic receptors and also with biochemical studies on an alpha-Bgt affinity purified locust AChR. Thus, a functional receptor exhibiting the characteristic pharmacology of an in vivo insect nicotinic AChR can be expressed in Xenopus oocytes by injection with a single subunit RNA. PMID:1702381

  3. Mechanisms of acetylcholine receptor loss in myasthenia gravis.

    PubMed Central

    Drachman, D B; Adams, R N; Stanley, E F; Pestronk, A

    1980-01-01

    The fundamental abnormality affecting the neuromuscular junctions of myasthenic patients is a reduction of available AChRs, due to an autoimmune attack directed against the receptors. Antibodies to AChR are present in most patients, and there is evidence that they have a predominant pathogenic role in the disease, aided by complement. The mechanism of antibody action involves acceleration of the rate of degradation of AChRs, attributable to cross-linking of the receptors. In addition, antibodies may block AChRs, and may participate in producing destructive changes, perhaps in conjunction with complement. The possibility that cell-mediated mechanisms may play a role in the autoimmune responses of some myasthenic patients remains to be explored. Although the target of the autoimmune attack in myasthenic patients is probably always the acetylcholine receptors, it is not yet clear which of these immune mechanisms are most important. It is likely that the relative role of each mechanism varies from patient to patient. One of the goals of future research will be to identify the relative importance of each of these mechanisms in the individual patient, and to tailor specific immunotherapeutic measures to the abnormalities found. PMID:6249894

  4. 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.

    PubMed

    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

  5. Cholinergic modulation of the medial prefrontal cortex: the role of nicotinic receptors in attention and regulation of neuronal activity

    PubMed Central

    Bloem, Bernard; Poorthuis, Rogier B.; Mansvelder, Huibert D.

    2014-01-01

    Acetylcholine (ACh) release in the medial prefrontal cortex (mPFC) is crucial for normal cognitive performance. Despite the fact that many have studied how ACh affects neuronal processing in the mPFC and thereby influences attention behavior, there is still a lot unknown about how this occurs. Here we will review the evidence that cholinergic modulation of the mPFC plays a role in attention and we will summarize the current knowledge about the role between ACh receptors (AChRs) and behavior and how ACh receptor activation changes processing in the cortical microcircuitry. Recent evidence implicates fast phasic release of ACh in cue detection and attention. This review will focus mainly on the fast ionotropic nicotinic receptors and less on the metabotropic muscarinic receptors. Finally, we will review limitations of the existing studies and address how innovative technologies might push the field forward in order to gain understanding into the relation between ACh, neuronal activity and behavior. PMID:24653678

  6. Designing Human m1 Muscarinic Receptor-Targeted Hydrophobic Eigenmode Matched Peptides as Functional Modulators

    PubMed Central

    Selz, Karen A.; Mandell, Arnold J.; Shlesinger, Michael F.; Arcuragi, Vani; Owens, Michael J.

    2004-01-01

    A new proprietary de novo peptide design technique generated ten 15-residue peptides targeting and containing the leading nontransmembrane hydrophobic autocorrelation wavelengths, “modes”, of the human m1 muscarinic cholinergic receptor, m1AChR. These modes were also shared by the m4AChR subtype (but not the m2, m3, or m5 subtypes) and the three-finger snake toxins that pseudoirreversibly bind m1AChR. The linear decomposition of the hydrophobically transformed m1AChR amino acid sequence yielded ordered eigenvectors of orthogonal hydrophobic variational patterns. The weighted sum of two eigenvectors formed the peptide design template. Amino acids were iteratively assigned to template positions randomly, within hydrophobic groups. One peptide demonstrated significant functional indirect agonist activity, and five produced significant positive allosteric modulation of atropine-reversible, direct-agonist-induced cellular activation in stably m1AChR-transfected Chinese hamster ovary cells, reflected in integrated extracellular acidification responses. The peptide positive allosteric ligands produced left-shifts and peptide concentration-response augmentation in integrated extracellular acidification response asymptotic sigmoidal functions and concentration-response behavior in Hill number indices of positive cooperativity. Peptide mode specificity was suggested by negative crossover experiments with human m2ACh and D2 dopamine receptors. Morlet wavelet transformation of the leading eigenvector-derived, m1AChR eigenfunctions locates seven hydrophobic transmembrane segments and suggests possible extracellular loop locations for the peptide-receptor mode-matched, modulatory hydrophobic aggregation sites. PMID:14990463

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

    PubMed

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

    2012-08-01

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

  8. Characterization of the retina in the alpha7 nicotinic acetylcholine receptor knockout mouse

    NASA Astrophysics Data System (ADS)

    Smith, Marci L.

    Acetylcholine receptors (AChRs) are involved in visual processing and are expressed by inner retinal neurons in all species studied to date (Keyser et al., 2000; Dmitrieva et al., 2007; Liu et al., 2009), but their distribution in the mouse retina remains unknown. Reductions in alpha7 nicotinic AChRs (nAChRs) are thought to contribute to memory and visual deficits observed in Alzheimer's and schizophrenia (Coyle et al., 1983; Nordberg et al., 1999; Leonard et al., 2006). However, the alpha7 nAChR knockout (KO) mouse has a mild phenotype (Paylor et al., 1998; Fernandes et al., 2006; Young et al., 2007; Origlia et al., 2012). The purpose of this study was to determine the expression of AChRs in wildtype (WT) mouse retina and to assess whether up-regulation of other AChRs in the alpha7 nAChR KO retina may explain the minimal deficits described in the KO mouse. Reverse-transcriptase PCR (RT-PCR) showed that mRNA transcripts for alpha2-7, alpha 9, alpha10, beta2-4 nAChR subunits and m1-m5 muscarinic AChR (mAChR) subtypes were present in WT murine retina. Western blot analysis confirmed the presence of alpha3-5, alpha9, and m1-m5 AChR proteins and immunohistochemical analysis demonstrated nAChR and mAChR proteins expressed by subsets of bipolar, amacrine and ganglion cells. This is the first reported expression of alpha9 and alpha10 nAChR transcripts and alpha9 nAChR proteins in the retina of any species. Quantitative RT-PCR (qPCR) showed changes in AChR transcript expression in the alpha7 nAChR KO mouse retina relative to WT. Within whole retina alpha2, alpha9, alpha10, beta4, m1 and m4 AChR transcripts were up-regulated, while alpha5 nAChR transcripts were down-regulated. However, cell populations showed subtle differences; m4 mAChR transcripts were up-regulated in the ganglion cell layer and outer portion of the inner nuclear layer (oINL),while beta4 nAChR transcript up-regulation was limited to the oINL. Surprisingly, alpha2, alpha9, beta4, m2 and m4 transcripts were

  9. Prenatal restraint stress decreases the expression of alpha-7 nicotinic receptor in the brain of adult rat offspring.

    PubMed

    Baier, Carlos J; Pallarés, María E; Adrover, Ezequiela; Monteleone, Melisa C; Brocco, Marcela A; Barrantes, Francisco J; Antonelli, Marta C

    2015-01-01

    Prenatal stress (PS) strongly impacts fetal brain development and function in adulthood. In normal aging and Alzheimer's disease, there is hypothalamic-pituitary-adrenal axis dysfunction and loss of cholinergic neurons and neuronal nicotinic acetylcholine receptors (nAChRs). This study investigated whether prenatal restraint stress affects nAChR expression in the brain of adult offspring. For PS, pregnant dams were placed in a plastic restrainer for 45 min, three times daily during the last week of pregnancy; controls were undisturbed. Male offspring were analyzed at postnatal day (PND) 60 (n = 4 rats per group). Western blot (WB) and fluorescence microscopy showed that PS decreased α7-AChR subunit expression (∼50%) in the frontal cortex in the adult offspring. PS decreased significantly the number of α7-AChR-expressing cells in the medial prefrontal cortex (by ∼25%) and in the sensory-motor cortex (by ∼20%) without affecting the total cell number in those areas. No alterations were found in the hippocampus by quantitative polymerase chain reaction (qPCR), or WB analysis, but a detailed fluorescence microscopy analysis showed that PS affected α7-AChR mainly in the CA3 and dentate gyrus subfields: PS decreased α7-AChR subunit expression by ∼25 and ∼30%, respectively. Importantly, PS decreased the number of α7-AChR-expressing cells and the total cell number (by ∼15 and 20%, respectively) in the dentate gyrus. PS differently affected α4-AChR: PS impaired its mRNA expression in the frontal cortex (by ∼50%), without affecting protein levels. These results demonstrate that disturbances during gestation produce long-term alterations in the expression pattern of α7-AChR in rat brain. PMID:25798813

  10. Expression of m1-type muscarinic acetylcholine receptors by parvalbumin-immunoreactive neurons in the primary visual cortex: a comparative study of rat, guinea pig, ferret, macaque, and human.

    PubMed

    Disney, Anita A; Reynolds, John H

    2014-04-01

    Cholinergic neuromodulation is a candidate mechanism for aspects of arousal and attention in mammals. We have reported previously that cholinergic modulation in the primary visual cortex (V1) of the macaque monkey is strongly targeted toward GABAergic interneurons, and in particular that the vast majority of parvalbumin-immunoreactive (PV) neurons in macaque V1 express the m1-type (pirenzepine-sensitive, Gq-coupled) muscarinic ACh receptor (m1AChR). In contrast, previous physiological data indicates that PV neurons in rats rarely express pirenzepine-sensitive muscarinic AChRs. To examine further this apparent species difference in the cholinergic effectors for the primary visual cortex, we have conducted a comparative study of the expression of m1AChRs by PV neurons in V1 of rats, guinea pigs, ferrets, macaques, and humans. We visualize PV- and mAChR-immunoreactive somata by dual-immunofluorescence confocal microscopy and find that the species differences are profound; the vast majority (>75%) of PV-ir neurons in macaques, humans, and guinea pigs express m1AChRs. In contrast, in rats only ∼25% of the PV population is immunoreactive for m1AChRs. Our data reveal that while they do so much less frequently than in primates, PV neurons in rats do express Gq-coupled muscarinic AChRs, which appear to have gone undetected in the previous in vitro studies. Data such as these are critical in determining the species that represent adequate models for the capacity of the cholinergic system to modulate inhibition in the primate cortex. PMID:23983014

  11. Genetic Reconstitution of Functional Acetylcholine Receptor Channels in Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Claudio, Toni; Green, W. N.; Hartman, Deborah S.; Hayden, Deborah; Paulson, Henry L.; Sigworth, F. J.; Sine, Steven M.; Swedlund, Anne

    1987-12-01

    Foreign genes can be stably integrated into the genome of a cell by means of DNA-mediated gene transfer techniques, and large quantities of homogenous cells that continuously express these gene products can then be isolated. Such an expression system can be used to study the functional consequences of introducing specific mutations into genes and to study the expressed protein in the absence of cellular components with which it is normally in contact. All four Torpedo acetylcholine receptor (AChR) subunit complementary DNA's were introduced into the genome of a mouse fibroblast cell by DNA-mediated gene transfer. A clonal cell line that stably produced high concentrations of correctly assembled cell surface AChR's and formed proper ligand-gated ion channels was isolated. With this new expression system, recombinant DNA, biochemical, pharmacological, and electrophysiological techniques were combined to study Torpedo AChR's in a single intact system. The physiological and pharmacological profiles of Torpedo AChR's expressed in mouse fibroblast cells differ in some details from those described earlier, and may provide a more accurate reflection of the properties of this receptor in its natural environment.

  12. The linoleic acid derivative DCP-LA increases membrane surface localization of the α7 ACh receptor in a protein 4.1N-dependent manner.

    PubMed

    Kanno, Takeshi; Tsuchiya, Ayako; Tanaka, Akito; Nishizaki, Tomoyuki

    2013-03-01

    In yeast two-hybrid screening, protein 4.1N, a scaffolding protein, was identified as a binding partner of the α7 ACh (acetylcholine) receptor. For rat hippocampal slices, the linoleic acid derivative DCP-LA {8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid} increased the association of the α7 ACh receptor with 4.1N, and the effect was inhibited by GF109203X, an inhibitor of PKC (protein kinase C), although DCP-LA did not induce PKC phosphorylation of 4.1N. For PC-12 cells, the presence of the α7 ACh receptor in the plasma membrane fraction was significantly suppressed by knocking down 4.1N. DCP-LA increased the presence of the α7 ACh receptor in the plasma membrane fraction, and the effect was still inhibited by knocking down 4.1N. In the monitoring of α7 ACh receptor mobilization, DCP-LA enhanced signal intensities for the α7 ACh receptor at the membrane surface in PC-12 cells, which was clearly prevented by knocking down 4.1N. Taken together, the results of the present study show that 4.1N interacts with the α7 ACh receptor and participates in the receptor tethering to the plasma membrane. The results also indicate that DCP-LA increases membrane surface localization of the α7 ACh receptor in a 4.1N-dependent manner under the control of PKC, but without phosphorylating 4.1N. PMID:23256752

  13. The dual-acting H3 receptor antagonist and AChE inhibitor UW-MD-71 dose-dependently enhances memory retrieval and reverses dizocilpine-induced memory impairment in rats.

    PubMed

    Khan, Nadia; Saad, Ali; Nurulain, Syed M; Darras, Fouad H; Decker, Michael; Sadek, Bassem

    2016-01-15

    Both the histamine H3 receptor (H3R) and acetylcholine esterase (AChE) are involved in the regulation of release and metabolism of acetylcholine and several other central neurotransmitters. Therefore, dual-active H3R antagonists and AChE inhibitors (AChEIs) have shown in several studies to hold promise to treat cognitive disorders like Alzheimer's disease (AD). The novel dual-acting H3R antagonist and AChEI 7-(3-(piperidin-1-yl)propoxy)-1,2,3,9-tetrahydropyrrolo[2,1-b]quinazoline (UW-MD-71) with excellent selectivity profiles over both the three other HRs as well as the AChE's isoenzyme butyrylcholinesterase (BChE) shows high and balanced in vitro affinities at both H3R and AChE with IC50 of 33.9nM and hH3R antagonism with Ki of 76.2nM, respectively. In the present study, the effects of UW-MD-71 (1.25-5mg/kg, i.p.) on acquisition, consolidation, and retrieval in a one-trial inhibitory avoidance task in male rats were investigated applying donepezil (DOZ) and pitolisant (PIT) as reference drugs. Furthermore, the effects of UW-MD-71 on memory deficits induced by the non-competitive N-methyl-d-aspartate (NMDA) antagonist dizocilpine (DIZ) were tested. Our results indicate that administration of UW-MD-71 before the test session dose-dependently increased performance and enhanced procognitive effect on retrieval. However neither pre- nor post-training acute systemic administration of UW-MD-71 facilitated acquisition or consolidation. More importantly, UW-MD-71 (2.5mg/kg, i.p.) ameliorated the DIZ-induced amnesic effects. Furthermore, the procognitive activity of UW-MD-71 in retrieval was completely reversed and partly abrogated in DIZ-induced amnesia when rats were pretreated with the centrally-acting H2R antagonist zolantidine (ZOL), but not with the CNS penetrant H1R antagonist pyrilamine (PYR). These results demonstrate the procognitive effects of UW-MD-71 in two in vivo memory models, and are to our knowledge the first demonstration in vivo that a potent dual

  14. Expression of a Drosophila melanogaster acetylcholine receptor-related gene in the central nervous system

    SciTech Connect

    Wadsworth, S.C.; Rosenthal, L.S.; Kammermeyer, K.L.; Potter, M.B.; Nelson, D.J.

    1988-02-01

    The authors isolated Drosophila melanogaster genomic sequences with nucleotide and amino acid sequence homology to subunits of vertebrate acetylcholine receptor by hybridization with a Torpedo acetylcholine receptor subunit cDNA probe. Five introns are present in the portion of the Drosophila gene encoding the unprocessed protein and are positionally conserved relative to the human acetylcholine receptor alpha-subunit gene. The Drosophila genomic clone hybridized to salivary gland polytene chromosome 3L within region 64B and was termed AChR64B. A 3-kilobasae poly(A)-containing transcript complementary to the AChR64B clone was readily detectable by RNA blot hybridizations during midembryogenesis, during metamorphosis, and in newly enclosed adults. AChR64B transcripts were localized to the cellular regions of the central nervous system during embryonic, larval, pupal, and adult stages of development. During metamorphosis, a temporal relationship between the morphogenesis of the optic lobe and expression of AChR64B transcripts was observed.

  15. Association between Anti-Ganglionic Nicotinic Acetylcholine Receptor (gAChR) Antibodies and HLA-DRB1 Alleles in the Japanese Population

    PubMed Central

    Maeda, Yasuhiro; Migita, Kiyoshi; Higuchi, Osamu; Mukaino, Akihiro; Furukawa, Hiroshi; Komori, Atsumasa; Nakamura, Minoru; Hashimoto, Satoru; Nagaoka, Shinya; Abiru, Seigo; Yatsuhashi, Hiroshi; Matsuo, Hidenori; Kawakami, Atsushi; Yasunami, Michio; Nakane, Shunya

    2016-01-01

    Background/Aims Anti-ganglionic nicotinic acetylcholine receptor (gAChR) antibodies are observed in autoimmune diseases, as well as in patients with autoimmune autonomic ganglionopathy. However, the genetic background of anti-gAChR antibodies is unclear. Here, we investigated HLA alleles in autoimmune hepatitis (AIH) patients with or without anti-gAChR antibodies. Methodology/Principal Findings Genomic DNA from 260 patients with type-1 autoimmune hepatitis (AIH) were genotyped for HLA-A, B, DRB1, and DQB1 loci. Anti-gAChR antibodies in the sera form AIH patients were measured using the luciferase immunoprecipitation system, and examined allelic association in patients with or without anti-gAChR antibodies. Methodology/ Methods We detected anti-α3 or -β4 gAChR antibodies in 11.5% (30/260) of patients with AIH. Among AIH patients there was no significant association between HLA-A, B DQB1 alleles and the positivity for anti-gAChR antibodies. Whereas the HLA-DRB1*0403 allele showed a significantly increased frequency in AIH patients with anti-gAChR antibodies compared with those without anti-gAChR antibodies. Conclusions/Significance The frequency of the HLA-DRB1*0403 allele differed among Japanese patients with AIH according to the presence or absence of anti-gAChR antibodies. Our findings suggest that particular HLA class II molecules might control the development of anti-gAChR antibodies in the autoimmune response to gAChR. PMID:26807576

  16. Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

    PubMed Central

    Schwimmbeck, P L; Dyrberg, T; Drachman, D B; Oldstone, M B

    1989-01-01

    The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetylcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetylcholine receptor (HuAChR) alpha-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR alpha-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of alpha-bungarotoxin to the receptor. The HuAChR alpha-subunit 160-167 peptide demonstrated specific immunological cross-reactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR alpha-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia. Images PMID:2551924

  17. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    NASA Astrophysics Data System (ADS)

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-04-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ~30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs.

  18. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors

    PubMed Central

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  19. Structural correlates of affinity in fetal versus adult endplate nicotinic receptors.

    PubMed

    Nayak, Tapan Kumar; Chakraborty, Srirupa; Zheng, Wenjun; Auerbach, Anthony

    2016-01-01

    Adult-type nicotinic acetylcholine receptors (AChRs) mediate signalling at mature neuromuscular junctions and fetal-type AChRs are necessary for proper synapse development. Each AChR has two neurotransmitter binding sites located at the interface of a principal and a complementary subunit. Although all agonist binding sites have the same core of five aromatic amino acids, the fetal site has ∼30-fold higher affinity for the neurotransmitter ACh. Here we use molecular dynamics simulations of adult versus fetal homology models to identify complementary-subunit residues near the core that influence affinity, and use single-channel electrophysiology to corroborate the results. Four residues in combination determine adult versus fetal affinity. Simulations suggest that at lower-affinity sites, one of these unsettles the core directly and the others (in loop E) increase backbone flexibility to unlock a key, complementary tryptophan from the core. Swapping only four amino acids is necessary and sufficient to exchange function between adult and fetal AChRs. PMID:27101778

  20. N-Ethylmaleimide Dissociates α7 ACh Receptor from a Complex with NSF and Promotes Its Delivery to the Presynaptic Membrane.

    PubMed

    Nishizaki, Tomoyuki

    2016-08-01

    N-Ethylmaleimide (NEM)-sensitive factor (NSF) associates with soluble NSF attachment protein (SNAP), that binds to SNAP receptors (SNAREs) including syntaxin, SNAP25, and synaptobrevin. The complex of NSF/SNAP/SNAREs plays a critical role in the regulation of vesicular traffic. The present study investigated NEM-regulated α7 ACh receptor translocation. NSF associated with β-SNAP and the SNAREs syntaxin 1 and synaptobrevin 2 in the rat hippocampus. NSF also associated with the α7 ACh receptor subunit, the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1 and GluA2, and the γ-aminobutyric acid A (GABAA) receptor γ2 subunit. NEM, an inhibitor of NSF, significantly dissociated the α7 ACh receptor subunit from a complex with NSF and increased cell surface localization of the receptor subunit, but such effect was not obtained with the GluA1, GluA2 or γ2 subunits. NEM, alternatively, dissociated synaptobrevin 2 from an assembly of NSF/β-SNAP/syntaxin 1/synaptobrevin 2. NEM significantly increased the rate of nicotine-triggered AMPA receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, in rat hippocampal slices. The results of the present study indicate that NEM releases the α7 ACh receptor subunit and synaptobrevin 2 from an assembly of α7 ACh receptor subunit/NSF/β-SNAP/syntaxin 1/synaptobrevin 2, thereby promoting delivery of the α7 ACh receptor subunit to presynaptic membrane. PMID:27105867

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

    PubMed Central

    Verdiyan, Ekaterina E.; Allakhverdiev, Elvin S.; Maksimov, Georgy V.

    2016-01-01

    In the present paper we consider a new type of mechanism by which neurotransmitter acetylcholine (ACh) regulates the properties of peripheral nerve fibers myelin. Our data show the importance of the relationship between the changes in the number of Schwann cell (SC) acetylcholine receptors (AChRs) and the axon excitation (different intervals between action potentials (APs)). Using Raman spectroscopy, an effect of activation of SC AChRs on the myelin membrane fluidity was investigated. It was found, that ACh stimulates an increase in lipid ordering degree of the myelin lipids, thus providing evidence for specific role of the “axon-SC” interactions at the axon excitation. It was proposed, that during the axon excitation, the SC membrane K+- depolarization and the Ca2+—influx led to phospholipase activation or exocytosis of intracellular membrane vesicles and myelin structure reorganization. PMID:27455410

  2. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment

    PubMed Central

    Im, Sin-Hyeog; Barchan, Dora; Fuchs, Sara; Souroujon, Miriam C.

    1999-01-01

    Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR α-subunit (Hα1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Hα1-205–induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis. J. Clin. Invest. 104:1723–1730 (1999). PMID:10606626

  3. Suppression of ongoing experimental myasthenia by oral treatment with an acetylcholine receptor recombinant fragment.

    PubMed

    Im, S H; Barchan, D; Fuchs, S; Souroujon, M C

    1999-12-01

    Myasthenia gravis (MG) is an autoimmune disorder in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. In an attempt to develop an antigen-specific therapy for MG, we administered a nonmyasthenogenic recombinant fragment of AChR orally to rats. This fragment, corresponding to the extracellular domain of the human AChR alpha-subunit (Halpha1-205), protected rats from subsequently induced experimental autoimmune myasthenia gravis (EAMG) and suppressed ongoing EAMG when treatment was initiated during either the acute or chronic phases of disease. Prevention and suppression of EAMG were accompanied by a significant decrease in AChR-specific humoral and cellular responses. The underlying mechanism for the Halpha1-205-induced oral tolerance seems to be active suppression, mediated by a shift from a T-helper 1 (Th1) to a Th2/Th3 response. This shift was assessed by changes in the cytokine profile, a deviation of anti-AChR IgG isotypes from IgG2 to IgG1, and a suppressed AChR-specific delayed-type hypersensitivity response. Our results in experimental myasthenia suggest that oral administration of AChR-specific recombinant fragments may be considered for antigen-specific immunotherapy of myasthenia gravis. PMID:10606626

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

    PubMed

    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

  5. Functional and structural interaction of (-)-lobeline with human α4β2 and α4β4 nicotinic acetylcholine receptor subtypes.

    PubMed

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

    2015-07-01

    To determine the pharmacologic activity of (-)-lobeline between human (h)α4β2 and hα4β4 nicotinic acetylcholine receptors (AChRs), functional and structural experiments were performed. The Ca(2+) influx results established that (-)-lobeline neither actives nor enhances the function of the studied AChR subtypes, but competitively inhibits hα4β4 AChRs with potency ∼10-fold higher than that for hα4β2 AChRs. This difference is due to a higher binding affinity for the [(3)H]cytisine sites at hα4β4 compared to hα4β2 AChRs, which, in turn, can be explained by our molecular dynamics (MD) results: (1) higher stability of (-)-lobeline and its hydrogen bonds within the α4β4 pocket compared to the α4β2 pocket, (2) (-)-lobeline promotes Loop C to cap the binding site at the α4β4 pocket, but forces Loop C to get apart from the α4β2 pocket, precluding the gating process elicited by agonists, and (3) the orientation of (-)-lobeline within the α4β4, but not the α4β2, subpocket, promoted by the t- (or t+) rotameric state of α4-Tyr98, remains unchanged during the whole MD simulation. This study gives a detailed view of the molecular and dynamics events evoked by (-)-lobeline supporting the differential binding affinity and subsequent inhibitory potency between hα4β2 and hα4β4 AChRs, and supports the possibility that the latter subtype is also involved in its activity. PMID:25794424

  6. Nicotinic acetylcholine receptors (nAChRs) at zebrafish red and white muscle show different properties during development.

    PubMed

    Ahmed, Kazi T; Ali, Declan W

    2016-08-01

    Nicotinic acetylcholine receptors (nAChRs) are highly expressed at the vertebrate neuromuscular junction (NMJ) where they are required for muscle activation. Understanding the factors that underlie NMJ development is critical for a full understanding of muscle function. In this study we performed whole cell and outside-out patch clamp recordings, and single-cell RT-qPCR from zebrafish red and white muscle to examine the properties of nAChRs during the first 5 days of development. In red fibers miniature endplate currents (mEPCs) exhibit single exponential time courses at 1.5 days postfertilization (dpf) and double exponential time courses from 2 dpf onwards. In white fibers, mEPCs decay relatively slowly, with a single exponential component at 1.5 dpf. By 2 and 3 dpf, mEPC kinetics speed up, and decay with a double exponential component, and by 4 dpf the exponential decay reverts back to a single component. Single channel recordings confirm the presence of two main conductance classes of nAChRs (∼45 pS and ∼65 pS) in red fibers with multiple time courses. Two main conductance classes are also present in white fibers (∼55 pS and ∼73 pS), but they exhibit shorter mean open times by 5 dpf compared with red muscle. RT-qPCR of mRNA for nicotinic receptor subunits supports a switch from γ to ε subunits in white fibers but not in red. Our findings provide a developmental profile of mEPC properties from red and white fibers in embryonic and larval zebrafish, and reveal previously unknown differences between the NMJs of these muscle fibers.© 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 916-936, 2016. PMID:26585318

  7. T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

    PubMed Central

    Manfredi, A A; Protti, M P; Dalton, M W; Howard, J F; Conti-Tronconi, B M

    1993-01-01

    We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected. Images PMID:7688757

  8. Biochemical and functional properties of distinct nicotinic acetylcholine receptors in the superior cervical ganglion of mice with targeted deletions of nAChR subunit genes.

    PubMed

    David, Reinhard; Ciuraszkiewicz, Anna; Simeone, Xenia; Orr-Urtreger, Avi; Papke, Roger L; McIntosh, J M; Huck, Sigismund; Scholze, Petra

    2010-03-01

    Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in ganglia of the autonomic nervous system. Here, we determined the subunit composition of hetero-pentameric nAChRs in the mouse superior cervical ganglion (SCG), the function of distinct receptors (obtained by deletions of nAChR subunit genes) and mechanisms at the level of nAChRs that might compensate for the loss of subunits. As shown by immunoprecipitation and Western blots, wild-type (WT) mice expressed: alpha 3 beta 4 (55%), alpha 3 beta 4 alpha 5 (24%) and alpha 3 beta 4 beta 2 (21%) nAChRs. nAChRs in beta 4 knockout (KO) mice were reduced to < 15% of controls and no longer contained the alpha 5 subunit. Compound action potentials, recorded from the postganglionic (internal carotid) nerve and induced by preganglionic nerve stimulation, did not differ between alpha 5 beta 4 KO and WT mice, suggesting that the reduced number of receptors in the KO mice did not impair transganglionic transmission. Deletions of alpha 5 or beta2 did not affect the overall number of receptors and we found no evidence that the two subunits substitute for each other. In addition, dual KOs allowed us to study the functional properties of distinct alpha 3 beta4 and alpha 3 beta 2 receptors that have previously only been investigated in heterologous expression systems. The two receptors strikingly differed in the decay of macroscopic currents, the efficacy of cytisine, and their responses to the alpha-conotoxins AuIB and MII. Our data, based on biochemical and functional experiments and several mouse KO models, clarify and significantly extend previous observations on the function of nAChRs in heterologous systems and the SCG. PMID:20377613

  9. Calcium signalling mediated by the 9 acetylcholine receptor in a cochlear cell line from the immortomouse.

    PubMed

    Jagger, D J; Griesinger, C B; Rivolta, M N; Holley, M C; Ashmore, J F

    2000-08-15

    1. We have investigated the characteristics of the alpha9 acetylcholine receptor (alpha9AChR) expressed in hair cell precursors in an immortalized cell line UB/OC-2 developed from the organ of Corti of the transgenic H-2Kb-tsA58 mouse (the Immortomouse) using both calcium imaging and whole-cell recording. 2. Ratiometric measurements of fura-2 fluorescence revealed an increase of intracellular calcium concentration in cells when challenged with 10 microM ACh. The calcium increase was seen in 66 % of the cells grown at 39 degrees C in differentiated conditions. A sm aller fraction (34%) of cells grown at 33 degrees C in proliferative con ditions responded. 3. Caffeine (10mM) elevated cell calcium. In the ab sence of caffeine, the majority of imaged cells responded only once to A Ch presentations. Pretreatment with caffeine ingibited all calcium respo nses to ACh. 4. In whole-cell tight-seal recordings 10 microM ACh activa ted inward current was dependent on the extracellular calcium concentrat ion with an estimated PCa/PNa of 80 for the alpha9 receptor at physiological calcium levels. 5 . The data indicate that ACh activates a calcium-permeable channel alpha 9AChR in UB/OC-2 cells and that the channel has a significantly higher c alcium permeability than other AChRs. The results indicate that the alp ha9AChR may be able to elevate intracellular calcium levels in hair cell s both directly and via store release. PMID:11011664

  10. Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors

    PubMed Central

    Takahashi, Masazumi; Kubo, Tai; Mizoguchi, Akira; Carlson, C. George; Endo, Katsuaki; Ohnishi, Katsunori

    2002-01-01

    In mammals, two combinations of muscle nicotinic acetylcholine receptors (AChRs) are used: α2βγδ (γ-AChR) or α2βɛδ (ɛ-AChR). After birth, γ-AChRs are replaced by ɛ-AChRs (γ/ɛ-switch). The two receptors have different conductances and open times. During perinatal period, the long open time γ-AChRs generate random myofiber action potentials from uniquantal miniature end-plate potentials (mEPPs). ɛ-AChRs are suitable for strong adult muscle activities. Since the effect of the γ/ɛ-switch on neuromuscular development was unclear, despite the many differences in channel characteristics, we carried out this study to generate γ-subunit-deficient mice. Homozygotes born alive survived for 2 days in a stable condition, and were able to move their forelimbs. Endplate AChRs included ɛ-subunits, and muscle fibers had multiple neuromuscular junctions. Both pre- and postsynapses were abnormal and spontaneous action potentials generated from mEPPs were totally absent. Results suggest a requirement for γ-AChRs in mediating synaptically-induced action potential activity critical for neuromuscular development. PMID:12101101

  11. Amyloid-β peptide increases cell surface localization of α7 ACh receptor to protect neurons from amyloid β-induced damage.

    PubMed

    Jin, Yu; Tsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki

    Amyloid-β peptide 1-42 (Aβ1-42) reduced PC-12 cell viability in a concentration (1-10 μM)- and treatment time (48-72 h)-dependent manner. Nicotine prevented Aβ1-42-induced PC-12 cell death, but conversely, the α7 ACh receptor antagonist α-bungarotoxin enhanced Aβ1-42-induced cell toxicity. Extracellularly applied Aβ1-42 significantly increased cell surface localization of α7 ACh receptor in PC-12 cells as compared with that for non-treated control cells. Cell surface localization of α7 ACh receptor in the brain of 5xFAD mouse, an animal model of Alzheimer's disease (AD), apparently increased in an age (1-12 months)-dependent manner in association with increased accumulation of Aβ1-42 in the plasma membrane component. Taken together, these results indicate that Aβ1-42 promotes translocation of α7 ACh receptor towards the cell surface and that α7 ACh receptor rescues neuronal cells from Aβ1-42-induced damage. PMID:26522221

  12. END-PLATE ACETYLCHOLINE RECEPTOR: STRUCTURE, MECHANISM, PHARMACOLOGY, AND DISEASE

    PubMed Central

    Sine, Steven M.

    2012-01-01

    The synapse is a localized neurohumoral contact between a neuron and an effector cell and may be considered the quantum of fast intercellular communication. Analogously, the postsynaptic neurotransmitter receptor may be considered the quantum of fast chemical to electrical transduction. Our understanding of postsynaptic receptors began to develop about a hundred years ago with the demonstration that electrical stimulation of the vagus nerve released acetylcholine and slowed the heart beat. During the past 50 years, advances in understanding postsynaptic receptors increased at a rapid pace, owing largely to studies of the acetylcholine receptor (AChR) at the motor endplate. The endplate AChR belongs to a large superfamily of neurotransmitter receptors, called Cys-loop receptors, and has served as an exemplar receptor for probing fundamental structures and mechanisms that underlie fast synaptic transmission in the central and peripheral nervous systems. Recent studies provide an increasingly detailed picture of the structure of the AChR and the symphony of molecular motions that underpin its remarkably fast and efficient chemoelectrical transduction. PMID:22811427

  13. Muscle aches

    MedlinePlus

    ... common cause of muscle aches and pain is fibromyalgia , a condition that causes tenderness in your muscles ... imbalance, such as too little potassium or calcium Fibromyalgia Infections, including the flu, Lyme disease , malaria , muscle ...

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

    PubMed Central

    Li, Ben-Wen; Rush, Amy C.; Weil, Gary J.

    2015-01-01

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

  15. Chronic ethanol (EtOH) feeding increases muscarinic receptor (mAChR) density in esophagus without parallel change in dose response (D-R) to cholinergic agonists

    SciTech Connect

    Keshavarzian, A.; Gordon, J.H.; Urban, G.; Fields, J.Z. VA Hospital, Hines, IL )

    1991-03-11

    The mAChR/effector pathway for signal transduction is important in the physiology of esophagus and mAChR alterations are involved in EtOH induced changes in several organs. To see if EtOH-induced increases in lower esophageal sphincter pressure (LESP) are due to upregulation of mAChR, the authors evaluated mAChR binding and D-R curves for bethanechol (IV) induced increases in LESP, and compared these values to changes in LESP after acute and chronic EtOH. EtOH was given to cats acutely or chronically. The number of mAChR sites (Bmax) in esophagus was lowered by acute EtOH, withdrawal from chronic EtOH raised Bmax. Acute injection of EtOH to cats in withdrawal reversed this increase in mAChR density. These changes correlated with the earlier data on EtOH-induced changes in LESP. In contrast, the D-R curve for bethanechol shifted to the right. Thus, the withdrawal-associated increase in Bmax is more likely to be a compensatory response to deficits distal to the receptor recognition site than to proximal deficits and doesn't cause LESP hyperactivity. Also, receptor binding changes do not necessarily translate into physiological changes.

  16. Targeting the Nicotinic Acetylcholine Receptors (nAChRs) in Astrocytes as a Potential Therapeutic Target in Parkinson's Disease.

    PubMed

    Jurado-Coronel, Juan Camilo; Avila-Rodriguez, Marco; Capani, Francisco; Gonzalez, Janneth; Moran, Valentina Echeverria; Barreto, George E

    2016-01-01

    Parkinson's disease (PD) is a relatively common disorder of the Central Nervous System (CNS), whose etiology is characterized by a selective and progressive degeneration of dopaminergic neurons, and the presence of Lewy bodies in the pars compacta of the substantia nigra, and gaping dopamine depletion in the striatum. Patients with this disease suffer from tremors, slowness of movements, gait instability, and rigidity. These patients may also present functional disability, reduced quality of life, and rapid cognitive decline. It has been shown that nicotine exerts beneficial effects in patients with PD and in in-vitro and in-vivo models of this disease. Astrocytes are an important component in the immune response associated with PD, and that nicotine might be able to inhibit the inflammation-related apoptosis of these cells, being this a potential strategy for PD treatment. This action of nicotine could be due mainly to activation of α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells. However, nicotine administration can protect dopaminergic neurons against degeneration by inhibiting astrocytes activation in the substantia nigra pars compacta (SNpc) and therefore reduce inflammation. Owing to the toxicity and capacity of nicotine to induce addiction, analogues of this substance have been designed and tested in various experimental paradigms, and targeting α7-nAChRs expressed in glial cells may be a novel therapeutic strategy for PD treatment. PMID:26972289

  17. Antigenic role of single residues within the main immunogenic region of the nicotinic acetylcholine receptor.

    PubMed Central

    Papadouli, I; Potamianos, S; Hadjidakis, I; Bairaktari, E; Tsikaris, V; Sakarellos, C; Cung, M T; Marraud, M; Tzartos, S J

    1990-01-01

    The target of most of the autoantibodies against the acetylcholine receptor (AChR) in myasthenic sera is the main immunogenic region (MIR) on the extracellular side of the AChR alpha-subunit. Binding of anti-MIR monoclonal antibodies (mAbs) has been recently localized between residues alpha 67 and alpha 76 of Torpedo californica electric organ (WNPADYGGIK) and human muscle (WNPDDYGGVK) AChR. In order to evaluate the contribution of each residue to the antigenicity of the MIR, we synthesized peptides corresponding to residues alpha 67-76 from Torpedo and human AChRs, together with 13 peptide analogues. Nine of these analogues had one residue of the Torpedo decapeptide replaced by L-alanine, three had a structure which was intermediate between those of the Torpedo and human alpha 67-76 decapeptides, and one had D-alanine in position 73. Binding studies employing six anti-MIR mAbs and all 15 peptides revealed that some residues (Asn68 and Asp71) are indispensable for binding by all mAbs tested, whereas others are important only for binding by some mAbs. Antibody binding was mainly restricted to residues alpha 68-74, the most critical sequence being alpha 68-71. Fish electric organ and human MIR form two distinct groups of strongly overlapping epitopes. Some peptide analogues enhanced mAb binding compared with Torpedo and human peptides, suggesting that the construction of a very antigenic MIR is feasible. PMID:1695844

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

    PubMed

    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

  19. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed

    McCrea, P D; Popot, J L; Engelman, D M

    1987-12-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  20. Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

    PubMed Central

    McCrea, P D; Popot, J L; Engelman, D M

    1987-01-01

    Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit. PMID:3428268

  1. Synthesis and Biological Evaluation of Novel Carbon-11 Labeled Pyridyl Ethers: Candidate Ligands for In Vivo Imaging of α4β2 Nicotinic Acetylcholine Receptors (α4β2-nAChRs) in the brain with Positron Emission Tomography

    PubMed Central

    Gao, Yongjun; Ravert, Hayden T.; Kuwabara, Hiroto; Xiao, Yingxian; Endres, Christopher J.; Hilton, John; Holt, Daniel P.; Kumar, Anil; Alexander, Mohab; Wong, Dean F.; Dannals, Robert F.; Horti, Andrew G.

    2009-01-01

    The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics. PMID:19481945

  2. Muscarinic acetylcholine receptors are expressed by most parvalbumin-immunoreactive neurons in area MT of the macaque

    PubMed Central

    Disney, Anita A; Alasady, Hussein A; Reynolds, John H

    2014-01-01

    Background In the mammalian neocortex, cells that express parvalbumin (PV neurons) comprise a dominant class of inhibitory neuron that substantially overlaps with the fast/narrow-spiking physiological phenotype. Attention has pronounced effects on narrow-spiking neurons in the extrastriate cortex of macaques, and more consistently so than on their broad-spiking neighbors. Cortical neuromodulation by acetylcholine (ACh) is a candidate mechanism for aspects of attention and in the primary visual cortex (V1) of the macaque, receptors for ACh (AChRs) are strongly expressed by inhibitory neurons. In particular, most PV neurons in macaque V1 express m1 muscarinic AChRs and exogenously applied ACh can cause the release of γ-aminobutyric acid. In contrast, few PV neurons in rat V1 express m1 AChRs. While this could be a species difference, it has also been argued that macaque V1 is anatomically unique when compared with other cortical areas in macaques. Aims The aim of this study was to better understand the extent to which V1 offers a suitable model circuit for cholinergic anatomy in the macaque occipital lobe, and to explore cholinergic modulation as a biological basis for the changes in circuit behavior seen with attention. Materials and methods We compared expression of m1 AChRs by PV neurons between area V1 and the middle temporal visual area (MT) in macaque monkeys using dual-immunofluorescence confocal microscopy. Results and conclusion We find that, as in V1, most PV neurons in MT express m1 AChRs but, unlike in V1, it appears that so do most excitatory neurons. This provides support for V1 as a model of cholinergic modulation of inhibition in macaque visual cortex, but not of cholinergic modulation of visual cortical circuits in general. We also propose that ACh acting via m1 AChRs is a candidate underlying mechanism for the strong effects of attention on narrow-spiking neurons observed in behaving animals. PMID:24944872

  3. [18F]ASEM, a radiolabeled antagonist for imaging the α7-nicotinic acetylcholine receptor (α7-nAChR) with positron emission tomography (PET)

    PubMed Central

    Horti, Andrew G.; Gao, Yongjun; Kuwabara, Hiroto; Wang, Yuchuan; Abazyan, Sofya; Yasuda, Robert P.; Tran, Thao; Xiao, Yingxian; Sahibzada, Niaz; Holt, Daniel P.; Kellar, Kenneth J.; Pletnikov, Mikhail V.; Pomper, Martin G.; Wong, Dean F.; Dannals, Robert F.

    2014-01-01

    The α7-nicotinic cholinergic receptor (α7-nAChR) is a key mediator of brain communication and has been implicated in a wide variety of central nervous system disorders. None of the currently available PET radioligands for α7-nAChR are suitable for quantitative PET imaging, mostly due to insufficient specific binding. The goal of this study was to evaluate the potential of [18F]ASEM ([18F]JHU82132) as an α7-nAChR radioligand for PET. Methods Inhibition binding assay and receptor functional properties of ASEM were assessed in vitro. The brain regional distribution of [18F]ASEM in baseline and blockade were evaluated in DISC1 mice (dissection) and baboons (PET). Results ASEM is an antagonist for the α7-nAChR with high binding affinity (Ki = 0.3 nM). [18F]ASEM readily entered the baboon brain and specifically labeled α7-nAChR. The in vivo specific binding of [18F]ASEM in the brain regions enriched with α7-nAChRs was 80–90%. SSR180711, an α7-nAChR selective partial agonist, blocked [18F]ASEM binding in the baboon brain in a dose-dependent manner, suggesting that the binding of [18F]ASEM was mediated by α7-nAChRs and the radioligand was suitable for drug evaluation studies. In the baboon baseline studies, the brain regional volume of distribution (VT) values for [18F]ASEM were 23 (thalamus), 22 (insula), 18 (hippocampus) and 14 (cerebellum), whereas in the binding selectivity (blockade) scan, all regional VT values were reduced to less than 4. The range of regional binding potential (BPND) values in the baboon brain was from 3.9 to 6.6. In vivo cerebral binding of [18F]ASEM and α7-nAChR expression in mutant DISC1 mice, a rodent model of schizophrenia, was significantly lower than in control animals, which is in agreement with previous post-mortem human data. Conclusion [18F]ASEM holds promise as a radiotracer with suitable imaging properties for quantification of α7-nAChR in the human brain. PMID:24556591

  4. Muscle-Specific Receptor Tyrosine Kinase (MuSK) Myasthenia Gravis.

    PubMed

    Hurst, Rebecca L; Gooch, Clifton L

    2016-07-01

    Autoimmune myasthenia gravis (MG) is the prototypic, antibody-mediated neuromuscular disease and is characterized by a decrease in the number of functional acetylcholine receptors (AChR) within the muscle end plate zone of the neuromuscular junction (NMJ). Although the pathophysiology of AChR-mediated myasthenia gravis has been extensively studied over the last 40 years since its original description by Patrick and Lindstrom (Science 180:871-872, 1973), less is known about the much more recently described muscle-specific kinase (MuSK) antibody-mediated MG. MuSK-MG has features clinically distinct from Ach-R MG, as well as a different pattern of response to treatment and a unique immunopathogenesis. PMID:27170368

  5. Poly(Ethylene Glycol) as a Scaffold for High-Affinity Open-Channel Blockers of the Mouse Nicotinic Acetylcholine Receptor

    PubMed Central

    Lin, Wan-Chen; Licht, Stuart

    2014-01-01

    High-affinity blockers for an ion channel often have complex molecular structures that are synthetically challenging and/or laborious. Here we show that high-affinity blockers for the mouse nicotinic acetylcholine receptor (AChR) can be prepared from a structurally simple material, poly(ethylene glycol) (PEG). The PEG-based blockers (PQ1–5), comprised of a flexible octa(ethylene glycol) scaffold and two terminal quaternary ammonium groups, exert low- to sub-micromolar affinities for the open AChR pore (measured via single-channel analysis of AChRs expressed in human embryonic kidney cells). PQ1–5 are comparable in pore-binding affinity to the strongest AChR open-channel blockers previously reported, which have complex molecular structures. These results suggest a general approach for designing potent open-channel blockers from a structurally flexible polymer. This design strategy involves simple synthetic procedures and does not require detailed information about the structure of an ion-channel pore. PMID:25386750

  6. Photolabeling of membrane-bound Torpedo nicotinic acetylcholine receptor with the hydrophobic probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine

    SciTech Connect

    White, B.J.; Cohen, J.B.

    1988-11-29

    The hydrophobic, photoactivatable probe 3-trifluoromethyl-3-(m-(/sup 125/I)iodophenyl)diazirine ((/sup 125/I)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 ..gamma..-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 ..cap alpha..-bungarotoxin and the noncompetitive antagonist phencyclidine had only modest effect on (/sup 125/I)TID labeling of the AChR. The regions of the AChR ..cap alpha..-subunit that incorporate (/sup 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 (/sup 125/I)TID but in a carbamylcholine-insensitive manner. Two further cleavage fragments, which together span about one-third of the ..cap alpha..-subunit amino terminus, incorporated no detectable (/sup 125/I)TID. The mapping results place constraints on suggested models of AChR subunit topology.

  7. P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression

    PubMed Central

    Martinez, Camila Guerra; Zamith-Miranda, Daniel; da Silva, Marcia Gracindo; Ribeiro, Karla Consort; Brandão, Izaíra Trincani; Silva, Celio Lopes; Diaz, Bruno Lourenço; Bellio, Maria; Persechini, Pedro Muanis; Kurtenbach, Eleonora

    2015-01-01

    Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM. PMID:26592184

  8. In vivo pharmacological interactions between a type II positive allosteric modulator of α7 nicotinic ACh receptors and nicotinic agonists in a murine tonic pain model

    PubMed Central

    Freitas, K; Negus, SS; Carroll, FI; Damaj, MI

    2013-01-01

    Background and Purpose The α7 nicotinic ACh receptor subtype is abundantly expressed in the CNS and in the periphery. Recent evidence suggests that α7 nicotinic ACh receptor (nAChR) subtypes, which can be activated by an endogenous cholinergic tone comprising ACh and the α7 agonist choline, play an important role in chronic pain and inflammation. In this study, we evaluated whether type II α7 positive allosteric modulator PNU-120596 induces antinociception on its own and in combination with choline in the formalin pain model. Experimental Approach We assessed the effects of PNU-120596 and choline and the nature of their interactions in the formalin test using an isobolographic analysis. In addition, we evaluated the interaction of PNU-120596 with PHA-54613, an exogenous selective α7 nAChR agonist, in the formalin test. Finally, we assessed the interaction between PNU-120596 and nicotine using acute thermal pain, locomotor activity, body temperature and convulsing activity tests in mice. Key Results We found that PNU-120596 dose-dependently attenuated nociceptive behaviour in the formalin test after systemic administration in mice. In addition, mixtures of PNU-120596 and choline synergistically reduced formalin-induced pain. PNU-120596 enhanced the effects of nicotine and α7 agonist PHA-543613 in the same test. In contrast, PNU-120596 failed to enhance nicotine-induced convulsions, hypomotility and antinociception in acute pain models. Surprisingly, it enhanced nicotine-induced hypothermia via activation of α7 nAChRs. Conclusions and Implications Our results demonstrate that type II α7 positive allosteric modulators produce antinociceptive effects in the formalin test through a synergistic interaction with the endogenous α7 agonist choline. PMID:23004024

  9. Physiological characterization of human muscle acetylcholine receptors from ALS patients

    PubMed Central

    Palma, Eleonora; Inghilleri, Maurizio; Conti, Luca; Deflorio, Cristina; Frasca, Vittorio; Manteca, Alessia; Pichiorri, Floriana; Roseti, Cristina; Torchia, Gregorio; Limatola, Cristina; Grassi, Francesca; Miledi, Ricardo

    2011-01-01

    Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: “microtransplantation” of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease. PMID:22128328

  10. Modal gating of muscle nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Vij, Ridhima

    Many ion channels exhibit multiple patterns of kinetic activity in single-channel currents. This behavior is rare in WT mouse muscle nicotinic acetylcholine receptors (AChRs), where A2C↔A2O gating events are well-described by single exponentials. Also, single-channel open probability (PO) is essentially homogeneous at a given agonist concentration in the WT receptors. Here I report that perturbations of almost all the residues in loop C (alpha188-alpha199, at the agonist binding site) generate heterogeneity in PO ('modes'). Such unsettled activity was apparent with an alanine substitution at all positions in loop C (except alphaY190 and alphaY198) and with different side chain substitutions at alphaP197 for both adult- and fetal-type AChRs. I used single channel electrophysiology along with site-directed mutagenesis to study modal gating in AChRs consequent to mutations/deletions in loop C. The multiple patterns of kinetic activity arose from the difference in agonist affinity rather than in intrinsic AChR gating. Out of the four different agonists used to study the modal behavior, acetylcholine (ACh) showed a higher degree of kinetic heterogeneity compared to others. The time constant for switching between modes was long (~mins), suggesting that they arise from alternative, stable protein conformations. By studying AChRs having only 1 functional binding site, I attempted to find the source of the affinity difference, which was traced mainly to the alphadelta agonist site. Affinity at the neurotransmitter binding site is mainly determined by a core of five aromatic residues (alphaY93, alphaW149, alphaY190, alphaY198 and deltaW57). Phenylalanine substitutions at all aromatic residues except alphaY93 resulted in elimination of modes. Modes were also eliminated by alanine mutation at deltaW57 on the complementary side but not at other aromatics. Also, by substituting four gamma subunit residues into the delta subunit on the complementary beta sheet, I found that

  11. Identification of regions involved in the binding of alpha-bungarotoxin to the human alpha7 neuronal nicotinic acetylcholine receptor using synthetic peptides.

    PubMed Central

    Marinou, Martha; Tzartos, Socrates J

    2003-01-01

    The neuronal alpha7 nicotinic acetylcholine receptor (AChR) binds the neurotoxin alpha-bungarotoxin (alpha-Bgt). Fine mapping of the alpha-Bgt-binding site on the human alpha7 AChR was performed using synthetic peptides covering the entire extracellular domain of the human alpha7 subunit (residues 1-206). Screening of these peptides for (125)I-alpha-Bgt binding resulted in the identification of at least two toxin-binding sites, one at residues 186-197, which exhibited the best (125)I-alpha-Bgt binding, and one at residues 159-165, with weak toxin-binding capacity; these correspond, respectively, to loops C and IV of the agonist-binding site. Toxin binding to the alpha7(186-197) peptide was almost completely inhibited by unlabelled alpha-Bgt or d -tubocurarine. Alanine substitutions within the sequence 186-198 revealed a predominant contribution of aromatic and negatively charged residues to the binding site. This sequence is homologous to the alpha-Bgt binding site of the alpha1 subunit (residues 188-200 in Torpedo AChR). In competition experiments, the soluble peptides alpha7(186-197) and Torpedo alpha1(184-200) inhibited the binding of (125)I-alpha-Bgt to the immobilized alpha7(186-197) peptide, to native Torpedo AChR, and to the extracellular domain of the human alpha1 subunit. These results suggest that the toxin-binding sites of the neuronal alpha7 and muscle-type AChRs bind to identical or overlapping sites on the alpha-Bgt molecule. In support of this, when synthetic alpha-Bgt peptides were tested for binding to the recombinant extracellular domains of the human alpha7 and alpha1 subunits, and to native Torpedo and alpha7 AChR, the results indicated that alpha-Bgt interacts with both neuronal and muscle-type AChRs through its central loop II and C-terminal tail. PMID:12614199

  12. Further proof of the existence of a non-neuronal cholinergic system in the human Achilles tendon: Presence of the AChRα7 receptor in tendon cells and cells in the peritendinous tissue.

    PubMed

    Forsgren, Sture; Alfredson, Håkan; Andersson, Gustav

    2015-11-01

    Human tendon cells have the capacity for acetylcholine (ACh) production. It is not known if the tendon cells also have the potential for ACh breakdown, nor if they show expression of the nicotinic acetylcholine receptor AChRα7 (α7nAChR). Therefore, tendon tissue specimens from patients with midportion Achilles tendinopathy/tendinosis and from normal midportion Achilles tendons were examined. Reaction for the degradative enzyme acetylcholinesterase (AChE) was found in some tenocytes in only a few tendinopathy tendons, and was never found in those of control tendons. Tenocytes displayed more regularly α7nAChR immunoreactivity. However, there was a marked heterogeneity in the degree of this reaction within and between the specimens. α7nAChR immunoreactivity was especially pronounced for tenocytes showing an oval/widened appearance. There was a tendency that the magnitude of α7nAChR immunoreactivity was higher in tendinopathy tendons as compared to control tendons. A stronger α7nAChR immunoreactivity than seen for tenocytes was observed for the cells in the peritendinous tissue. It is likely that the α7nAChR may be an important part of an auto-and paracrine loop of non-neuronal ACh that is released from the tendon cells. The effects may be related to proliferative and blood vessel regulatory functions as well as features related to collagen deposition. ACh can furthermore be of importance in leading to anti-inflammatory effects in the peritendinous tissue, a tissue nowadays considered to be of great relevance for the tendinopathy process. Overall, the findings show that tendon tissue, a tissue known to be devoid of cholinergic innervation, is a tissue in which there is a marked non-neuronal cholinergic system. PMID:25981114

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

    SciTech Connect

    Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

    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.

  14. Mammalian 43-kD acetylcholine receptor-associated protein (RAPsyn) is expressed in some nonmuscle cells

    SciTech Connect

    Musil, L.S.; Frail, D.E.; Merlie, J.P. )

    1989-05-01

    Torpedo electric organ and vertebrate neuromuscular junctions contain the receptor-associated protein of the synapse (RAPsyn) (previously referred to as the 43K protein), a nonactin, 43,000-Mr peripheral membrane protein associated with the cytoplasmic face of postsynaptic membranes at areas of high nicotinic acetylcholine receptor (AChR) density. Although not directly demonstrated, several lines of evidence suggest that RAPsyn is involved in the synthesis and/or maintenance of such AChR clusters. Microscopic and biochemical studies had previously indicated that RAPsyn expression is restricted to differentiated, AChR-synthesizing cells. Our recent finding that RAPsyn is also produced in undifferentiated myocytes led to to examine whether RAPsyn is synthesized in cell types that never express AChR (i.e., cells of other than skeletal muscle origin). Various primary and established rodent cell lines were metabolically labeled with (35S)methionine, and extracts were immunoprecipitated with a monospecific anti-RAPsyn serum. Analysis of these immunoprecipitates by SDS-PAGE revealed detectable RAPsyn synthesis in some (notably fibroblast and Leydig tumor cell lines and primary cardiac cells) but not all (hepatocyte- and lymphocyte-derived) cell types. These results were further substantiated by peptide mapping studies of RAPsyn immunoprecipitated from different cells and quantitation of RAPsyn-encoding mRNA levels in mouse tissues. RAPsyn synthesized in both muscle and nonmuscle cells was shown to be tightly associated with membranes. These findings demonstrate that RAPsyn is not specific to skeletal muscle-derived cells and imply that it may function in a capacity either in addition to or instead of AChR clustering.

  15. Role of M2 domain residues in conductance and gating of acetylcholine receptors in developing Xenopus muscle

    PubMed Central

    Sullivan, Michael P; Owens, Jesse L; Kullberg, Richard W

    1999-01-01

    The contributions of specific residues in γ- and ɛ-subunits to the developmental changes in conductance and open time of Xenopus muscle acetylcholine receptors (AChRs) were investigated. This study was directed primarily at residues in the M2 domains of γ- and ɛ-subunits; however, the results of additional mutations in the extracellular region flanking M2 and in the amphipathic region between M3 and M4 are also described. The M2 domains of γ- and ɛ-subunits differ at only three amino acid residues, two of which are adjacent to each other and located near the narrowest part of the pore. These two residues (NI in γ, SV in ɛ) were found to be major determinants of the difference in conductance and open time of AChRs bearing γ- or ɛ-subunits. Mutation of N to S in the γ-subunit converted the long open time of receptors bearing the γ-subunit (γ-AChRs) to the brief open time characteristic of receptors bearing an ɛ-subunit (ɛ-AChRs). Conversely, ɛ-AChRs with SV mutated to NI in the ɛ-subunit exhibited a long open time characteristic of γ-AChRs. Mutation of N to S in the γ-subunit increased the conductance of γ-AChRs but did not confer the full conductance of wild-type ɛ-AChRs. Conversely, mutation of SV to NI in the ɛ-subunit reduced the conductance of ɛ-AChRs, but not completely to the level of wild-type γ-AChRs. PMID:9925875

  16. Development of radiohalogenated muscarinic ligands for the in vivo imaging of m-AChR by nuclear medicine techniques

    SciTech Connect

    McPherson, D.W.; Luo, H.; Knapp, F.F. Jr.

    1994-06-01

    Alterations in the density of acetylcholinergic muscarinic receptors (m-AChR) have been observed in various dementias. This has spurred interest in the development of radiohalogenated ligands which can be used for the non-invasive in vivo detection of m-AChR by nuclear medicine techniques. We have developed a new ligand 1-azabicyclo[2.2.2]oct-3-yl ({alpha}-hydroxy-{alpha}-(1-iodo-1-propen-3-yl)-{alpha}-phenylacetate (IQNP,12) which demonstrates high affinity for the muscarinic receptor. When labeled with radioiodine it has been shown to be selective and specific for m-ACHR. Initial studies on the separation and in vivo evaluation of the various isomers of IQNP have shown that the stereochemistry of the chiral centers and the configuration around the double bond play an important role in m-AChR subtype specificity. In vivo evaluation of these stereoisomers demonstrate that E-(R,R)-IQNP has a high affinity for the M{sub 1} muscarinic subtype while Z-(R,R)-IQNP demonstrate a high affinity for M{sub 1} and M{sub 2} receptor subtypes. These data demonstrate IQNP (12) has potential for use in the non-evasive in vivo detection of m-AChR by single photon emission computed tomography (SPECT). A brominated analogue, ``BrQNP,`` in which the iodine has been replaced by a bromine atom, has also been prepared and was shown to block the in vivo uptake of IQNP in the brain and heart and therefore has potential for positron emission tomographic (PET) studies of m-AChR.

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

    PubMed Central

    Hu, Hong-Zhen; Li, Zhi-Wang

    1997-01-01

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

  18. Behavioral phenotyping of heterozygous acetylcholinesterase knockout (AChE+/-) mice showed no memory enhancement but hyposensitivity to amnesic drugs.

    PubMed

    Espallergues, Julie; Galvan, Laurie; Sabatier, Florence; Rana-Poussine, Vanessa; Maurice, Tangui; Chatonnet, Arnaud

    2010-01-20

    Decrease in the expression or activity of acetylcholinesterase (AChE) enzymatic activity results in increased cholinergic tonus in the brain and periphery, with concomitant regulations of nicotinic and muscarinic receptors expression. We generated AChE knockout mice and characterized the behavioral phenotype of heterozygous animals, focusing on learning and memory functions. Male and female, AChE+/- and AChE+/+ littermate controls (129 sv strain) were tested at 5-9 weeks of age. AChE activity was significantly decreased in the hippocampus and cortex of AChE+/- mice, but butyrylcholinesterase activity was preserved. AChE+/- mice failed to show any difference in terms of locomotion, exploration and anxiety parameters in the open-field test. Animals were then tested for place learning in the water-maze. They were trained using a 'sustained acquisition' protocol (3 swim trials per day) or a 'mild acquisition' protocol (2 swim trials per day) to locate an invisible platform in fixed position (reference memory procedure). Then, during 3 days, they were trained to locate the platform in a variable position (working memory procedure). Learning profiles and probe test performances were similar for AChE+/- and AChE+/+ mice. Mice were then treated with the muscarinic receptor antagonist scopolamine (0.5, 5 mg/kg) 20 min before each training session. Scopolamine impaired learning at both doses in AChE+/+ mice, but only at the highest dose in AChE+/- mice. Moreover, the intracerebroventricular injection of amyloid-beta25-35 peptide, 9 nmol, 7 days before water-maze acquisition, failed to induce learning deficits in AChE+/- mice, but impaired learning in AChE+/+ controls. The peptide failed to be toxic in forebrain structures of AChE+/- mice, since an increase in lipid peroxidation levels was measured in the hippocampus of AChE+/+ but not AChE+/- mice. We conclude that the increase in cholinergic tonus observed in AChE+/- mice did not result in increased memory functions but

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

    PubMed

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

    2016-07-01

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

  20. A mutational analysis of the acetylcholine receptor channel transmitter binding site.

    PubMed Central

    Akk, G; Zhou, M; Auerbach, A

    1999-01-01

    Mutagenesis and single-channel kinetic analysis were used to investigate the roles of four acetylcholine receptor channel (AChR) residues that are candidates for interacting directly with the agonist. The EC50 of the ACh dose-response curve was increased following alpha-subunit mutations Y93F and Y198F and epsilon-subunit mutations D175N and E184Q. Single-channel kinetic modeling indicates that the increase was caused mainly by a reduced gating equilibrium constant (Theta) in alphaY198F and epsilonD175N, by an increase in the equilibrium dissociation constant for ACh (KD) and a reduction in Theta in alphaY93F, and only by a reduction in KD in epsilonE184Q. This mutation altered the affinity of only one of the two binding sites and was the only mutation that reduced competition by extracellular K+. Additional mutations of epsilonE184 showed that K+ competition was unaltered in epsilonE184D and was virtually eliminated in epsilonE184K, but that neither of these mutations altered the intrinsic affinity for ACh. Thus there is an apparent electrostatic interaction between the epsilonE184 side chain and K+ ( approximately 1.7kBT), but not ACh+. The results are discussed in terms of multisite and induced-fit models of ligand binding to the AChR. PMID:9876135

  1. A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model.

    PubMed

    Gattenlöhner, S; Jörissen, H; Huhn, M; Vincent, A; Beeson, D; Tzartos, S; Mamalaki, A; Etschmann, B; Muller-Hermelink, H K; Koscielniak, E; Barth, S; Marx, A

    2010-01-01

    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) gamma-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. PMID:20204062

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

    PubMed Central

    Gattenlöhner, S.; Jörißen, H.; Huhn, M.; Vincent, A.; Beeson, D.; Tzartos, S.; Mamalaki, A.; Etschmann, B.; Muller-Hermelink, H. K.; Koscielniak, E.; Barth, S.; Marx, A.

    2010-01-01

    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. PMID:20204062

  3. Cholinergic receptors as target for cancer therapy in a systems medicine perspective.

    PubMed

    Russo, P; Del Bufalo, A; Milic, M; Salinaro, G; Fini, M; Cesario, A

    2014-01-01

    Epithelial cells not innervated by cholinergic neurons express nicotinic and muscarinic acetylcholine (ACh) receptors (nAChR, mAChR). nAChR and mAChR are components of the auto-/paracrine-regulatory loop of non-neuronal ACh release. The cholinergic control of non-neuronal cells may be mediated by different effects (synergistic, additive, or reciprocal) triggered by these receptors. The ionic events (Ca(+2) influx) are generated by the ACh-opening of nAChR channels, while the metabolic events by ACh-binding to G-proteincoupled mAChR. Effective inter- and intracellular signaling is crucial for valuable cancer cells proliferation and survival. Depending on cancer cell type, different AChR have been identified. The proliferation of airways epithelial cancer cells and pancreatic cancer cells may be under the control of α7-nAChR and M3-mAChR, while breast cancer cells and colon cancer cells are regulated by α9-nAChR, and M3-mAChR, respectively. In turn, these receptors may activate different pathways (Ras-Raf-1-Erk-AKT) as well as other receptors (β- adrenergicR). nAChR or mAChR antagonists may inhibit cancer growth. Inhibition of M3 by antisense or antagonists (Darifenacin, Tiotropium) reduces lung or colon cancer proliferation, as well as inhibition of α9- nAChR [polyphenol (-)-epigallocatechin-3-gallate] diminishes breast cancer cells growth. α7-nAChR silencing inhibits lung cancer proliferation. Moreover, inhibition of the nAChR-β-adrenergicR pathway (β-blockers) could be also useful. This review will describe the future translational perspectives of cholinergic receptors druginhibition in a complex disease such as cancer that poses compelling treatment challenges. Cancer happens as consequence of disease-perturbed molecular networks in relevant organ cells that change during progression. The framework for approaching these challenges is a systems approach. PMID:25324001

  4. Natural killer cells and single nucleotide polymorphisms of specific ion channels and receptor genes in myalgic encephalomyelitis/chronic fatigue syndrome

    PubMed Central

    Marshall-Gradisnik, Sonya; Huth, Teilah; Chacko, Anu; Johnston, Samantha; Smith, Pete; Staines, Donald

    2016-01-01

    Aim The aim of this paper was to determine natural killer (NK) cytotoxic activity and if single nucleotide polymorphisms (SNPs) and genotypes in transient receptor potential (TRP) ion channels and acetylcholine receptors (AChRs) were present in isolated NK cells from previously identified myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS) patients. Subjects and methods A total of 39 ME/CFS patients (51.69±2 years old) and 30 unfatigued controls (47.60±2.39 years old) were included in this study. Patients were defined according to the 1994 Centers for Disease Control and Prevention criteria. Flow cytometry protocols were used to examine NK cytotoxic activity. A total of 678 SNPs from isolated NK cells were examined for 21 mammalian TRP ion channel genes and for nine mammalian AChR genes via the Agena Bioscience iPlex Gold assay. SNP association and genotype was determined using analysis of variance and Plink software. Results ME/CFS patients had a significant reduction in NK percentage lysis of target cells (17%±4.68%) compared with the unfatigued control group (31%±6.78%). Of the 678 SNPs examined, eleven SNPs for TRP ion channel genes (TRPC4, TRPC2, TRPM3, and TRPM8) were identified in the ME/CFS group. Five of these SNPs were associated with TRPM3, while the remainder were associated with TRPM8, TRPC2, and TRPC4 (P<0.05). Fourteen SNPs were associated with nicotinic and muscarinic AChR genes: six with CHRNA3, while the remainder were associated with CHRNA2, CHRNB4, CHRNA5, and CHRNE (P<0.05). There were sixteen genotypes identified from SNPs in TRP ion channels and AChRs for TRPM3 (n=5), TRPM8 (n=2), TRPC4 (n=3), TRPC2 (n=1), CHRNE (n=1), CHRNA2 (n=2), CHRNA3 (n=1), and CHRNB4 (n=1) (P<0.05). Conclusion We identified a number of SNPs and genotypes for TRP ion channels and AChRs from isolated NK cells in patients with ME/CFS, suggesting these SNPs and genotypes may be involved in changes in NK cell function and the development of ME/CFS pathology

  5. Docking studies of benzylidene anabaseine interactions with α7 nicotinic acetylcholine receptor (nAChR) and acetylcholine binding proteins (AChBPs): Application to the design of related α7 selective ligands

    PubMed Central

    Kombo, David C.; Mazurov, Anatoly; Tallapragada, Kartik; Hammond, Philip S.; Chewning, Joseph; Hauser, Terry A.; Vasquez-Valdivieso, Montserrat; Yohannes, Daniel; Talley, Todd T.; Taylor, Palmer; Caldwell, William S.

    2016-01-01

    AChBPs isolated from Lymnaea stagnalis (Ls), Aplysia californica (Ac) and Bulinus truncatus (Bt) have been extensively used as structural prototypes to understand the molecular mechanisms that underlie ligand-interactions with nAChRs [1]. Here, we describe docking studies on interactions of benzylidene anabaseine analogs with AChBPs and α7 nAChR. Results reveal that docking of these compounds using Glide software accurately reproduces experimentally-observed binding modes of DMXBA and of its active metabolite, in the binding pocket of Ac. In addition to the well-known nicotinic pharmacophore (positive charge, hydrogen-bond acceptor, and hydrophobic aromatic groups), a hydrogen-bond donor feature contributes to binding of these compounds to Ac, Bt, and the α7 nAChR. This is consistent with benzylidene anabaseine analogs with OH and NH2 functional groups showing the highest binding affinity of these congeners, and the position of the ligand shown in previous X-ray crystallographic studies of ligand-Ac complexes. In the predicted ligand-Ls complex, by contrast, the ligand OH group acts as hydrogen-bond acceptor. We have applied our structural findings to optimizing the design of novel spirodiazepine and spiroimidazoline quinuclidine series. Binding and functional studies revealed that these hydrogen-bond donor containing compounds exhibit improved affinity and selectivity for the α7 nAChR subtype and demonstrate partial agonism. The gain in affinity is also due to conformational restriction, tighter hydrophobic enclosures, and stronger cation-π interactions. The use of AChBPs structure as a surrogate to predict binding affinity to α7 nAChR has also been investigated. On the whole, we found that molecular docking into Ls binding site generally scores better than when a α7 homology model, Bt or Ac crystal structure is used. PMID:21986237

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

    PubMed Central

    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 (Cch) and to identify the MAchR subtype involved. ASMC were isolated from tracheal fragments of Sprague–Dawley rats by enzymatic digestion. Proliferation assays were performed by MTS-PMS method. Viability was confirmed by trypan blue exclusion method. Mitogens as, epidermal growth factor (EGF), Tumor necrosis factor-alpha (TNF-α) and fetal bovine serum (FBS) increased ASMC proliferation (p < 0.05, n = 5). Cch alone increased ASMC proliferation at 24 and 48 hrs. However, combination of Cch with other mitogens exhibited a dual effect, synergistic proliferation effect in the presence of EGF (5 ng/mL) and 5% FBS and inhibiting the proliferation induced by 10% FBS, EGF (10 ng/mL) and TNF-α (10 ng/mL). To determine the MAChR subtype involved in these biological responses, a titration curve of selective muscarinic antagonists were performed. The Cch stimulatory and inhibitory effects on ASCM proliferation was blocked by AF-DX-116 (M2AChR selective antagonist), in greater proportion than 4-DAMP (M3AChR selective antagonist), suggesting that the modulation of muscarinic agonist-induced proliferation is M2AChR mediated responses. Thus, M2AChR can activate multiple signal transduction systems and mediate both effects on ASMC proliferation depending on the plethora and variable airway microenvironments existing in asthma and COPD. PMID:24377382

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

    PubMed

    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

  8. Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.

    PubMed Central

    Yu, L; LaPolla, R J; Davidson, N

    1986-01-01

    Clones coding for the mouse nicotinic acetylcholine receptor (AChR) gamma subunit precursor have been selected from a cDNA library derived from a mouse myogenic cell line and sequenced. The deduced protein sequence consists of a signal peptide of 22 amino acid residues and a mature gamma subunit of 497 amino acid residues. There is a high degree of sequence conservation between this mouse sequence and published human and calf AChR gamma subunits and, after allowing for functional amino acid substitutions, also to the more distantly related chicken and Torpedo AChR gamma subunits. The degree of sequence conservation is especially high in the four putative hydrophobic membrane spanning regions, supporting the assignment of these domains. RNA blot hybridization showed that the mRNA level of the gamma subunit increases by 30 fold or more upon differentiation of the two mouse myogenic cell lines, BC3H-1 and C2C12, suggesting that the primary controls for changes in gene expression during differentiation are at the level of transcription. One cDNA clone was found to correspond to a partially processed nuclear transcript containing two as yet unspliced intervening sequences. Images PMID:3010242

  9. O-fucosylation of muscle agrin determines its ability to cluster acetylcholine receptors

    PubMed Central

    Kim, Mi-Lyang; Chandrasekharan, Kumaran; Glass, Matthew; Shi, Shaolin; Stahl, Mark C.; Kaspar, Brian; Stanley, Pamela

    2008-01-01

    Protein O-fucosyltransferase 1 (Pofut1) transfers fucose to serine or threonine on proteins, including Notch receptors, that contain EGF repeats with a particular consensus sequence. Here we demonstrate that agrin is O-fucosylated in a Pofut1-dependent manner, and that this glycosylation can regulate agrin function. Fucosylation of recombinant C45 agrin, both active (neural, z8) and inactive (muscle, z0) splice forms, was eliminated when agrin was overexpressed in Pofut1-deficient cells or by mutation of a consensus site for Pofut1 fucosylation (serine 1726 in the EGF4 domain). Loss of O-fucosylation caused a gain of function for muscle agrin such that it stimulated AChR clustering and MuSK phosphorylation in cultured myotubes at levels normally only found with the neural splice form. Deletion of Pofut1 in cultured primary myotubes and in adult skeletal muscle increased AChR aggregation. In addition, Pofut1 gene and protein expression and Pofut1 activity of the EGF4 domain of agrin were modulated during neuromuscular development. These data are consistent with a role for Pofut1 in AChR aggregation during synaptogenesis via the regulation of the synaptogenic activity of muscle agrin. PMID:18775496

  10. The Association of PTPN22 R620W Polymorphism Is Stronger with Late-Onset AChR-Myasthenia Gravis in Turkey

    PubMed Central

    Kaya, Gizem A.; Coşkun, Ayse N.; Yılmaz, Vuslat; Oflazer, Piraye; Gülsen-Parman, Yeşim; Aysal, Fikret; Disci, Rian; Direskeneli, Haner; Marx, Alexander; Deymeer, Feza; Saruhan-Direskeneli, Güher

    2014-01-01

    A functional single nucleotide polymorphism (SNP) of the PTPN22 gene encoding a protein tyrosine phosphatase has been associated with autoimmune disorders including myasthenia gravis (MG). As the PTPN22 R620W polymorphism has a wide variation of allele frequencies among different populations, this polymorphism was investigated in MG in Turkey. An emphasis is put on MG subgroups according to autoantibody (Abs) production and presence of thymoma. DNA samples from 416 patients with clinically diagnosed generalized MG (231 with Abs to acetylcholine receptor, AChR-MG), 53 with Abs to muscle-specific kinase (MuSK-MG), 55 patients with no detectable Abs (SN-MG), 77 patients with thymoma (TAMG) and 293 healthy controls (HC) were genotyped for the SNP (PTPN22 R620W, C1858T, rs2476601). The PTPN22 T allele was increased in AChR-MG patients (odds ratio [OR]: 2.5, 95%CI: 1.2–5.1). The association was stronger in late disease-onset AChR (LOMG, OR: 3.1, 95%CI: 1.2–8.2). MuSK-MG, SN-MG and TAMG groups did not carry the variant allele more frequently than the HC. In contrast to findings in other autoimmune diseases, the distribution of the PTPN22 polymorphism in this population provides a susceptibility marker for AChR-MG. The strongest association is detected in patients with LOMG. PMID:25119822

  11. Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

    PubMed Central

    Machold, J; Utkin, Y; Kirsch, D; Kaufmann, R; Tsetlin, V; Hucho, F

    1995-01-01

    A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis. Images Fig. 2 PMID:7543679

  12. Antidepressant activity in mice elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, a positive allosteric modulator of the α7 nicotinic acetylcholine receptor.

    PubMed

    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

  13. Energy for Wild-Type Acetylcholine Receptor Channel Gating from Different Choline Derivatives

    PubMed Central

    Bruhova, Iva; Gregg, Timothy; Auerbach, Anthony

    2013-01-01

    Agonists, including the neurotransmitter acetylcholine (ACh), bind at two sites in the neuromuscular ACh receptor channel (AChR) to promote a reversible, global change in protein conformation that regulates the flow of ions across the muscle cell membrane. In the synaptic cleft, ACh is hydrolyzed to acetate and choline. Replacement of the transmitter’s ester acetyl group with a hydroxyl (ACh→choline) results in a +1.8 kcal/mol reduction in the energy for gating generated by each agonist molecule from a low- to high-affinity change of the transmitter binding site (ΔGB). To understand the distinct actions of structurally related agonist molecules, we measured ΔGB for 10 related choline derivatives. Replacing the hydroxyl group of choline with different substituents, such as hydrogen, chloride, methyl, or amine, increased the energy for gating (i.e., it made ΔGB more negative relative to choline). Extending the ethyl hydroxide tail of choline to propyl and butyl hydroxide also increased this energy. Our findings reveal the amount of energy that is available for the AChR conformational change provided by different, structurally related agonists. We speculate that a hydrogen bond between the choline hydroxyl and the backbone carbonyl of αW149 positions this agonist’s quaternary ammonium group so as to reduce the cation-π interaction between this moiety and the aromatic groups at the binding site. PMID:23442907

  14. Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

    SciTech Connect

    Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.; McIntosh, J. Michael; Han, Kyou-Hoon . E-mail: khhan600@kribb.re.kr

    2006-06-23

    {alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

  15. New potential AChE inhibitor candidates.

    PubMed

    de Paula, A A N; Martins, J B L; dos Santos, M L; Nascente, L de C; Romeiro, L A S; Areas, T F M A; Vieira, K S T; Gambôa, N F; Castro, N G; Gargano, R

    2009-09-01

    We have theoretically studied new potential candidates of acetylcholinesterase (AChE) inhibitors designed from cardanol, a non-isoprenoid phenolic lipid of cashew Anacardium occidentale nut-shell liquid. The electronic structure calculations of fifteen molecule derivatives from cardanol were performed using B3LYP level with 6-31G, 6-31G(d), and 6-311+G(2d,p) basis functions. For this study we used the following groups: methyl, acetyl, N,N-dimethylcarbamoyl, N,N-dimethylamine, N,N-diethylamine, piperidine, pyrrolidine, and N,N-methylbenzylamine. Among the proposed compounds we identified that the structures with substitution by N,N-dimethycarbamoyl, N,N-dimethylamine, and pyrrolidine groups were better correlated to rivastigmine, and represent possible AChE inhibitors against Alzheimer disease. PMID:19446931

  16. Polymerase chain reaction-based diagnosis of rhabdomyosarcomas: comparison of fetal type acetylcholine receptor subunits and myogenin.

    PubMed

    Gattenlöhner, S; Müller-Hermelink, H K; Marx, A

    1998-06-01

    The diagnosis of rhabdomyosarcoma (RMS) is usually straight-forward when light microscopy and immunohistochemistry are used. However, tumors that exhibit a low degree of differentiation and small biopsies can lead to confusion. In such patients and for the detection of minimal (residual) disease, a polymerase chain reaction (PCR)-based approach would be a valuable diagnostic adjunct. This type of approach would be highly sensitive and should be free from the risk for contamination of the tumor sample with normal tissue. Because myogenin and the alpha and gamma subunit of the fetal type acetylcholine receptor (AChR) are specific immunohistochemical markers for RMS, their expression on the mRNA level in RMS, other childhood and adult tumors, and normal tissues was studied. Although the sensitivity of both approaches was 100% in embryonal and alveolar RMS, detection of myogenin mRNA was not specific for RMS but occurred in normal muscle and the majority of the other normal tissues and childhood tumors. Conversely, detection of fetal AChR mRNA as defined by an alpha/tau ratio of < 1 was encountered only in RMS and denervated muscle. The authors conclude that mRNA of the fetal type AChR but not myogenin is a highly specific and sensitive target for the PCR-based diagnosis of RMS. PMID:9836066

  17. Activation of muscarinic receptors by ACh release in hippocampal CA1 depolarizes VIP but has varying effects on parvalbumin-expressing basket cells

    PubMed Central

    Bell, L Andrew; Bell, Karen A; McQuiston, A Rory

    2015-01-01

    We investigated the effect of acetylcholine release on mouse hippocampal CA1 perisomatically projecting interneurons. Acetylcholine was optogenetically released in hippocampal slices by expressing the excitatory optogenetic protein oChIEF-tdTomato in medial septum/diagonal band of Broca cholinergic neurons using Cre recombinase-dependent adeno-associated virally mediated transfection. The effect of optogenetically released acetylcholine was assessed on interneurons expressing Cre recombinase in vasoactive intestinal peptide (VIP) or parvalbumin (PV) interneurons using whole cell patch clamp methods. Acetylcholine released onto VIP interneurons that innervate pyramidal neuron perisomatic regions (basket cells, BCs) were depolarized by muscarinic receptors. Although PV BCs were also excited by muscarinic receptor activation, they more frequently responded with hyperpolarizing or biphasic responses. Muscarinic receptor activation resulting from ACh release increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in downstream hippocampal CA1 pyramidal neurons with peak instantaneous frequencies occurring in both the gamma and theta bandwidths. Both PV and VIP BCs contributed to the increased sIPSC frequency in pyramidal neurons and optogenetic suppression of PV or VIP BCs inhibited sIPSCs occurring in the gamma range. Therefore, we propose acetylcholine release in CA1 has a complex effect on CA1 pyramidal neuron output through varying effects on perisomatically projecting interneurons. PMID:25556796

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

    PubMed

    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

  19. Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

    PubMed Central

    Schloss, P; Hermans-Borgmeyer, I; Betz, H; Gundelfinger, E D

    1988-01-01

    The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65-212), the other domain is exposed to the cytoplasm (amino acids 305-444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha-bungarotoxin (alpha-Btx) binding sites occurring in Drosophila. Two different high-affinity binding sites for [125I]alpha-Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic-cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha-Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha-Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic-cholinergic binding sites in the insect nervous system. PMID:3141150

  20. Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

    PubMed Central

    Moiola, L; Karachunski, P; Protti, M P; Howard, J F; Conti-Tronconi, B M

    1994-01-01

    We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls. They secreted IL-2, not IL-4, suggesting that they comprised T helper type 1 cells. The T lines from MG patients could be propagated for several months. Three lines were tested with purified bovine muscle AChR and cross-reacted well with this antigen. All T lines were tested with the individual synthetic peptides present in the pool corresponding to the beta subunit sequence. Several beta subunit peptide sequences were recognized. Each line had an individual pattern of peptides recognition, but three sequence regions (peptides beta 181-200, beta 271-290, and the overlapping peptides beta 316-335 and beta 331-350) were recognized by most MG lines. The beta subunit-specific T lines from controls could be propagated for < 5 wk. Each line recognized several peptides, which frequently included the immunodominant regions listed above. Images PMID:7510715

  1. 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.

    PubMed

    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

  2. Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

    NASA Technical Reports Server (NTRS)

    Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

    1987-01-01

    A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

  3. Antibodies against low-density lipoprotein receptor-related protein 4 induce myasthenia gravis.

    PubMed

    Shen, Chengyong; Lu, Yisheng; Zhang, Bin; Figueiredo, Dwight; Bean, Jonathan; Jung, Jiung; Wu, Haitao; Barik, Arnab; Yin, Dong-Min; Xiong, Wen-Cheng; Mei, Lin

    2013-12-01

    Myasthenia gravis (MG) is the most common disorder affecting the neuromuscular junction (NMJ). MG is frequently caused by autoantibodies against acetylcholine receptor (AChR) and a kinase critical for NMJ formation, MuSK; however, a proportion of MG patients are double-negative for anti-AChR and anti-MuSK antibodies. Recent studies in these subjects have identified autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), an agrin receptor also critical for NMJ formation. LRP4 autoantibodies have not previously been implicated in MG pathogenesis. Here we demonstrate that mice immunized with the extracellular domain of LRP4 generated anti-LRP4 antibodies and exhibited MG-associated symptoms, including muscle weakness, reduced compound muscle action potentials (CMAPs), and compromised neuromuscular transmission. Additionally, fragmented and distorted NMJs were evident at both the light microscopic and electron microscopic levels. We found that anti-LRP4 sera decreased cell surface LRP4 levels, inhibited agrin-induced MuSK activation and AChR clustering, and activated complements, revealing potential pathophysiological mechanisms. To further confirm the pathogenicity of LRP4 antibodies, we transferred IgGs purified from LRP4-immunized rabbits into naive mice and found that they exhibited MG-like symptoms, including reduced CMAP and impaired neuromuscular transmission. Together, these data demonstrate that LRP4 autoantibodies induce MG and that LRP4 contributes to NMJ maintenance in adulthood. PMID:24200689

  4. Nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induce cyclooxygenase-2 activity in human gastric cancer cells: Involvement of nicotinic acetylcholine receptor (nAChR) and {beta}-adrenergic receptor signaling pathways

    SciTech Connect

    Shin, Vivian Yvonne; Jin, H.C.; Ng, Enders K.O.; Yu Jun; Leung, W.K.; Cho, C.H.; Sung, J.J.Y.

    2008-12-01

    Induction of cyclooxygenase-2 (COX-2) associates with cigarette smoke exposure in many malignancies. Nicotine and its derivative, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the two important components in cigarette smoke that contributes to cancer development. However, the molecular mechanism(s) by which nicotine or NNK promotes gastric carcinogenesis remains largely unknown. We found that nicotine and NNK significantly enhanced cell proliferation in AGS cells that expressed both alpha7 nicotinic acetylcholine receptor ({alpha}7 nAChR) and {beta}-adrenergic receptors. Treatment of cells with {alpha}-bungarotoxin ({alpha}-BTX, {alpha}7nAChR antagonist) or propranolol ({beta}-adrenergic receptor antagonist) blocked NNK-induced COX-2/PGE{sub 2} and cell proliferation, while nicotine-mediated cell growth and COX-2/PGE{sub 2} induction can only be suppressed by propranolol, but not {alpha}-BTX. Moreover, in contrast to the dependence of growth promoting effect of nicotine on Erk activation, inhibitor of p38 mitogen-activated protein kinase (MAPK) repressed NNK-induced COX-2 upregulation and resulted in suppression of cell growth. In addition, nicotine and NNK mediated COX-2 induction via different receptors to modulate several G1/S transition regulatory proteins and promote gastric cancer cell growth. Selective COX-2 inhibitor (SC-236) caused G1 arrest and abrogated nicotine/NNK-induced cell proliferation. Aberrant expression of cyclin D1 and other G1 regulatory proteins are reversed by blockade of COX-2. These results pointed to the importance of adrenergic and nicotinic receptors in gastric tumor growth through MAPK/COX-2 activation, which may perhaps provide a chemoprevention strategy for cigarette smoke-related gastric carcinogenesis.

  5. Analysis of acetylcholine receptor phosphorylation sites using antibodies to synthetic peptides and monoclonal antibodies.

    PubMed Central

    Safran, A; Neumann, D; Fuchs, S

    1986-01-01

    Three peptides corresponding to residues 354-367, 364-374, 373-387 of the acetylcholine receptor (AChR) delta subunit were synthesized. These peptides represent the proposed phosphorylation sites of the cAMP-dependent protein kinase, the tyrosine-specific protein kinase and the calcium/phospholipid-dependent protein kinase respectively. Using these peptides as substrates for phosphorylation by the catalytic subunit of cAMP-dependent protein kinase it was shown that only peptides 354-367 was phosphorylated whereas the other two were not. These results verify the location of the cAMP-dependent protein kinase phosphorylation site within the AChR delta subunit. Antibodies elicited against these peptides reacted with the delta subunit. The antipeptide antibodies and two monoclonal antibodies (7F2, 5.46) specific for the delta subunit were tested for their binding to non-phosphorylated receptor and to receptor phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. Antibodies to peptide 354-367 were found to react preferentially with non-phosphorylated receptor whereas the two other anti-peptide antibodies bound equally to phosphorylated and non-phosphorylated receptors. Monoclonal antibody 7F2 reacted preferentially with the phosphorylated form of the receptor whereas monoclonal antibody 5.46 did not distinguish between the two forms. Images Fig. 2. Fig. 4. Fig. 5. PMID:3816758

  6. Frizzled-9 impairs acetylcholine receptor clustering in skeletal muscle cells

    PubMed Central

    Avilés, Evelyn C.; Pinto, Cristina; Hanna, Patricia; Ojeda, Jorge; Pérez, Viviana; De Ferrari, Giancarlo V.; Zamorano, Pedro; Albistur, Miguel; Sandoval, Daniel; Henríquez, Juan P.

    2014-01-01

    Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain- and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ. PMID:24860427

  7. Role of L- and N-type Ca2+ channels in muscarinic receptor-mediated facilitation of ACh and noradrenaline release in the rat urinary bladder.

    PubMed Central

    Somogyi, G T; Zernova, G V; Tanowitz, M; de Groat, W C

    1997-01-01

    1. 3H-Noradrenaline (NA) and 14C-acetylcholine (ACh) released by electrical field stimulation were measured simultaneously in strips from the body of rat urinary bladder. 2. omega-Conotoxin GVIA (omega-CgTX; 20-100 nM) suppressed the non-facilitated transmitter release evoked by intermittent stimulation (IS), whereas nifedipine (1 microM) did not affect release. 3. Continuous electrical stimulation (CS) facilitated NA and ACh release via an atropine-sensitive mechanism. omega-CgTX reduced the facilitated release of NA (44% depression) but did not affect ACh release. Nifedipine depressed ACh release (43%) but not NA release. Combined administration of nifedipine and omega-CgTX (20 nM) produced a greater suppression of NA and ACh release (86 and 91%, respectively). 4. Maximal muscarinic facilitation of NA (5-fold) and ACh (17-fold) release occurred following administration of eserine, an anticholinesterase agent. Release of both NA and ACh was depressed by nifedipine (70 and 83%, respectively) but not by omega-CgTX. Combined application of omega-CgTX and nifedipine elicited a further depression of NA (95%) but not ACh release. 5. When NA and ACh release was facilitated with phorbol dibutyrate (0.5 microM), nifedipine inhibited ACh (67%) but not NA release, whereas omega-CgTX inhibited NA (73%) but not ACh release. Combined administration of both Ca2+ channel blockers did not elicit greater inhibition. 6. Bay K 8644, the L-type Ca2+ channel activator, increased ACh release in a dose-dependent manner (up to 5-fold) but did not significantly change NA release. 7. Both omega-CgTX (20-100 nM) and nifedipine (100 nM-1 microM) significantly decreased (50-80%) the neurally evoked contractions of the bladder strips. 8. It is concluded that L-type Ca2+ channels play a major role in muscarinic facilitation of NA and ACh release in the urinary bladder but are not essential for non-facilitated release. Other types of Ca2+ channels, including N-type, are involved to varying

  8. Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

    PubMed Central

    Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

    2014-01-01

    Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

  9. Functional differences between neurotransmitter binding sites of muscle acetylcholine receptors

    PubMed Central

    Nayak, Tapan K.; Bruhova, Iva; Chakraborty, Srirupa; Gupta, Shaweta; Zheng, Wenjun; Auerbach, Anthony

    2014-01-01

    A muscle acetylcholine receptor (AChR) has two neurotransmitter binding sites located in the extracellular domain, at αδ and either αε (adult) or αγ (fetal) subunit interfaces. We used single-channel electrophysiology to measure the effects of mutations of five conserved aromatic residues at each site with regard to their contribution to the difference in free energy of agonist binding to active versus resting receptors (ΔGB1). The two binding sites behave independently in both adult and fetal AChRs. For four different agonists, including ACh and choline, ΔGB1 is ∼−2 kcal/mol more favorable at αγ compared with at αε and αδ. Only three of the aromatics contribute significantly to ΔGB1 at the adult sites (αY190, αY198, and αW149), but all five do so at αγ (as well as αY93 and γW55). γW55 makes a particularly large contribution only at αγ that is coupled energetically to those contributions of some of the α-subunit aromatics. The hydroxyl and benzene groups of loop C residues αY190 and αY198 behave similarly with regard to ΔGB1 at all three kinds of site. ACh binding energies estimated from molecular dynamics simulations are consistent with experimental values from electrophysiology and suggest that the αγ site is more compact, better organized, and less dynamic than αε and αδ. We speculate that the different sensitivities of the fetal αγ site versus the adult αε and αδ sites to choline and ACh are important for the proper maturation and function of the neuromuscular synapse. PMID:25422413

  10. Decremental response to high-frequency trains of acetylcholine pulses but unaltered fractional Ca2+ currents in a panel of "slow-channel syndrome" nicotinic receptor mutants.

    PubMed

    Elenes, Sergio; Decker, Michael; Cymes, Gisela D; Grosman, Claudio

    2009-02-01

    The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (alphaS269I, betaV266M, epsilonL221F, epsilonT264P, and epsilonL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel-based approach to estimate the contribution of Ca(2+) to the total cation currents, we also found that none of these mutants affects the Ca(2+)-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca(2+)-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na(+), 1.8 mM Ca(2+), and 1.7 mM Mg(2+) on the extracellular side of cell-attached patches turned out be in the 5.0-9.4 pS range, representing a fractional Ca

  11. Muscarinic Acetylcholine Receptors in Macaque V1 Are Most Frequently Expressed by Parvalbumin-Immunoreactive Neurons

    PubMed Central

    Disney, Anita A.; Aoki, Chiye

    2010-01-01

    Acetylcholine (ACh) is believed to underlie mechanisms of arousal and attention in mammals. ACh also has a demonstrated functional effect in visual cortex that is both diverse and profound. We have reported previously that cholinergic modulation in V1 of the macaque monkey is strongly targeted toward GABAergic interneurons. Here we examine the localization of m1 and m2 muscarinic receptor subtypes across subpopulations of GABAergic interneurons—identified by their expression of the calcium-binding proteins parvalbumin, calbindin, and calretinin—using dual-immunofluorescence confocal microscopy in V1 of the macaque monkey. In doing so, we find that the vast majority (87%) of parvalbumin-immunoreactive neurons express m1-type muscarinic ACh receptors. m1 receptors are also expressed by 60% of calbindin-immunoreactive neurons and 40% of calretinin-immunoreactive neurons. m2 AChRs, on the other hand, are expressed by only 31% of parvalbumin neurons, 23% of calbindin neurons, and 25% of calretinin neurons. Parvalbumin-immunoreactive cells comprise ≈75% of the inhibitory neuronal population in V1 and included in this large subpopulation are neurons known to veto and regulate the synchrony of principal cell spiking. Through the expression of m1 ACh receptors on nearly all of these PV cells, the cholinergic system avails itself of powerful control of information flow through and processing within the network of principal cells in the cortical circuit. PMID:18265004

  12. Parazoanthoxanthin A blocks Torpedo nicotinic acetylcholine receptors.

    PubMed

    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

  13. Flow Cytofluorimetric Analysis of Anti-LRP4 (LDL Receptor-Related Protein 4) Autoantibodies in Italian Patients with Myasthenia Gravis

    PubMed Central

    Marino, Mariapaola; Scuderi, Flavia; Samengo, Daniela; Saltelli, Giorgia; Maiuri, Maria Teresa; Shen, Chengyong; Mei, Lin; Sabatelli, Mario; Pani, Giovambattista; Antonini, Giovanni; Evoli, Amelia; Bartoccioni, Emanuela

    2015-01-01

    Background Myasthenia gravis (MG) is an autoimmune disease in which 90% of patients have autoantibodies against the muscle nicotinic acetylcholine receptor (AChR), while autoantibodies to muscle-specific tyrosine kinase (MuSK) have been detected in half (5%) of the remaining 10%. Recently, the low-density lipoprotein receptor-related protein 4 (LRP4), identified as the agrin receptor, has been recognized as a third autoimmune target in a significant portion of the double sero-negative (dSN) myasthenic individuals, with variable frequency depending on different methods and origin countries of the tested population. There is also convincing experimental evidence that anti-LRP4 autoantibodies may cause MG. Methods The aim of this study was to test the presence and diagnostic significance of anti-LRP4 autoantibodies in an Italian population of 101 myasthenic patients (55 dSN, 23 AChR positive and 23 MuSK positive), 45 healthy blood donors and 40 patients with other neurological diseases as controls. All sera were analyzed by a cell-based antigen assay employing LRP4-transfected HEK293T cells, along with a flow cytofluorimetric detection system. Results We found a 14.5% (8/55) frequency of positivity in the dSN-MG group and a 13% frequency of co-occurrence (3/23) in both AChR and MuSK positive patients; moreover, we report a younger female prevalence with a mild form of disease in LRP4-positive dSN-MG individuals. Conclusion Our data confirm LRP4 as a new autoimmune target, supporting the value of including anti-LRP4 antibodies in further studies on Myasthenia gravis. PMID:26284792

  14. Acetylcholinesterase (AChE) inhibition aggravates fasting-induced triglyceride accumulation in the mouse liver.

    PubMed

    Yokota, Shin-Ichi; Nakamura, Kaai; Ando, Midori; Kamei, Hiroyasu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Shibata, Shigenobu

    2014-01-01

    Although fasting induces hepatic triglyceride (TG) accumulation in both rodents and humans, little is known about the underlying mechanism. Because parasympathetic nervous system activity tends to attenuate the secretion of very-low-density-lipoprotein-triglyceride (VLDL-TG) and increase TG stores in the liver, and serum cholinesterase activity is elevated in fatty liver disease, the inhibition of the parasympathetic neurotransmitter acetylcholinesterase (AChE) may have some influence on hepatic lipid metabolism. To assess the influence of AChE inhibition on lipid metabolism, the effect of physostigmine, an AChE inhibitor, on fasting-induced increase in liver TG was investigated in mice. In comparison with ad libitum-fed mice, 30 h fasting increased liver TG accumulation accompanied by a downregulation of sterol regulatory element-binding protein 1 (SREBP-1) and liver-fatty acid binding-protein (L-FABP). Physostigmine promoted the 30 h fasting-induced increase in liver TG levels in a dose-dependent manner, accompanied by a significant fall in plasma insulin levels, without a fall in plasma TG. Furthermore, physostigmine significantly attenuated the fasting-induced decrease of both mRNA and protein levels of SREBP-1 and L-FABP, and increased IRS-2 protein levels in the liver. The muscarinic receptor antagonist atropine blocked these effects of physostigmine on liver TG, serum insulin, and hepatic protein levels of SREBP-1 and L-FABP. These results demonstrate that AChE inhibition facilitated fasting-induced TG accumulation with up regulation of the hepatic L-FABP and SREBP-1 in mice, at least in part via the activation of muscarinic acetylcholine receptors. Our studies highlight the crucial role of parasympathetic regulation in fasting-induced TG accumulation, and may be an important source of information on the mechanism of hepatic disorders of lipid metabolism. PMID:25383314

  15. Modal affinities of endplate acetylcholine receptors caused by loop C mutations

    PubMed Central

    Vij, Ridhima; Purohit, Prasad

    2015-01-01

    The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity. PMID:26503719

  16. Coronaridine congeners inhibit human α3β4 nicotinic acetylcholine receptors by interacting with luminal and non-luminal sites.

    PubMed

    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

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

    PubMed

    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

  18. Increased efflux of amyloid-β peptides through the blood-brain barrier by muscarinic acetylcholine receptor inhibition reduces pathological phenotypes in mouse models of brain amyloidosis.

    PubMed

    Paganetti, Paolo; Antoniello, Katia; Devraj, Kavi; Toni, Nicolas; Kieran, Dairin; Madani, Rime; Pihlgren, Maria; Adolfsson, Oskar; Froestl, Wolfgang; Schrattenholz, André; Liebner, Stefan; Havas, Daniel; Windisch, Manfred; Cirrito, John R; Pfeifer, Andrea; Muhs, Andreas

    2014-01-01

    The formation and accumulation of toxic amyloid-β peptides (Aβ) in the brain may drive the pathogenesis of Alzheimer's disease. Accordingly, disease-modifying therapies for Alzheimer's disease and related disorders could result from treatments regulating Aβ homeostasis. Examples are the inhibition of production, misfolding, and accumulation of Aβ or the enhancement of its clearance. Here we show that oral treatment with ACI-91 (Pirenzepine) dose-dependently reduced brain Aβ burden in AβPPPS1, hAβPPSL, and AβPP/PS1 transgenic mice. A possible mechanism of action of ACI-91 may occur through selective inhibition of muscarinic acetylcholine receptors (AChR) on endothelial cells of brain microvessels and enhanced Aβ peptide clearance across the blood-brain barrier. One month treatment with ACI-91 increased the clearance of intrathecally-injected Aβ in plaque-bearing mice. ACI-91 also accelerated the clearance of brain-injected Aβ in blood and peripheral tissues by favoring its urinal excretion. A single oral dose of ACI-91 reduced the half-life of interstitial Aβ peptide in pre-plaque mhAβPP/PS1d mice. By extending our studies to an in vitro model, we showed that muscarinic AChR inhibition by ACI-91 and Darifenacin augmented the capacity of differentiated endothelial monolayers for active transport of Aβ peptide. Finally, ACI-91 was found to consistently affect, in vitro and in vivo, the expression of endothelial cell genes involved in Aβ transport across the Blood Brain Brain (BBB). Thus increased Aβ clearance through the BBB may contribute to reduced Aβ burden and associated phenotypes. Inhibition of muscarinic AChR restricted to the periphery may present a therapeutic advantage as it avoids adverse central cholinergic effects. PMID:24072071

  19. Structure-function studies of the muscle nicotinic acetylcholine receptor by site-directed mutagenesis in the pore region

    NASA Astrophysics Data System (ADS)

    Zhang, Haiyun

    In nicotinic acetylcholine receptors (nAChRs), as in glycine, GABA A, serotonin 5-HT3, and GluCl glutamate receptors, a leucine residue at the approximate midpoint (the 9' position) of the M2 transmembrane domain is conserved across all known subunits. We expressed the embryonic mouse muscle nAChRs with varying numbers (m* s) of subunits (2 αs, 1 β, 1 γ, and 1 δ) mutated at this position in Xenopus oocytes and discovered that mutations to serine (Leu9'Ser) result in a tenfold higher receptor sensitivity to acetylcholine (ACh) for each subunit mutated. Moreover, increases of side-chain polarity increase the sensitivity to ACh when other natural and unnatural residues are incorporated into this position. The data also indicated an especially strong interaction between the γ and δ subunits in the pore region, suggesting a specific arrangement of subunits within the pentamer. Detailed single-channel kinetic studies reveal that Leu9'Ser AChRs have (1) longer voltage- relaxation time constants, (2) longer ACh-induced openings and bursts, and (3) more frequent spontaneous openings. These effects increase with m* s. Synthesized postsynaptic currents were produced with a piezoelectric micromanipulator that delivered brief ACh pulses to multi-channel patches. Their decay time constants were, as expected, similar to the channel burst duration. Thus, both longer and more frequent openings contribute to the >=104-fold increase in the receptor sensitivity to ACh from the wild-type receptor to the receptor with m*s=4; and the highly conserved 9' leucine is crucial for the brief synaptic events that are normally observed. We also explored the effects of ligand-binding domain mutations: γD174N and δD180N (aspartic acid (D) to asparagine (N)). Macroscopic dose-response relations revealed that these mutations decrease the receptor's sensitivity to ACh. The combined effect with Leu9'Ser, however, differs from that predicted from a linear or independent sum of effects from

  20. Muscarinic receptor subtype determines vulnerability to oxidative stress in COS-7 cells.

    PubMed

    Joseph, J A; Fisher, D R; Strain, J

    2002-01-15

    Research has suggested that there may be increased brain-region selective vulnerability to oxidative stress in aging and that Vulnerability to oxidative stress may be important in determining regional differences in neuronal aging. We assessed whether one factor determining vulnerability to oxidative stress might involve qualitative/quantitative differences in receptor subtypes in various neuronal populations. COS-7 cells were transfected with one of five muscarinic receptor subtypes (M1-M5 AChR) to DA (1 mM for 4 h) and intracellular Ca2+ levels were examined via fluorescent imaging analysis prior to and following 750 microM oxotremorine (oxo). Results indicated that the ability of the cells to clear excess Ca2+ (i.e., Ca2+ Recovery) following oxo stimulation varied as a function of transfected mAChR subtype, with DA-treated M1, M2, or M4 cells showing greater decrements in Recovery than those transfected with M3 or M5 AChR. A similar pattern of results in M1- or M3-transfected DA-exposed cells was seen with respect to Viability. Viability of the untransfected cells was unaffected by DA. Pretreatment with Trolox (a Vitamin E analog) or PBN (a nitrone trapping agent) did not alter the DA effects on cell Recovery in the M1-transfected cells, but were effective in preventing the decrements in Viability. The calcium channel antagonists (L and N, respectively), Nifedipine and Conotoxin prevented both the DA-induced deficits in Recovery and Viability. Results are discussed in terms of receptor involvement in the regional differences in Vulnerability to oxidative stress with age, and that loss of neuronal function may not inevitably lead to cell death. PMID:11796204

  1. Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease.

    PubMed

    Hernandez, Ciria C; Nascimento, Jose H; Chaves, Elen A; Costa, Patricia C; Masuda, Masako O; Kurtenbach, Eleonora; Campos DE Carvalho, Antonio C; Gimenez, Luis E

    2008-01-01

    Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

  2. Autoantibodies Enhance Agonist Action and Binding to Cardiac Muscarinic Receptors in Chronic Chagas’ Disease

    PubMed Central

    Hernández, Ciria C.; Nascimento, José H.; Chaves, Elen A.; Costa, Patrícia C.; Masuda, Masako O.; Kurtenbach, Eleonora; Campos de Carvalho, Antônio C.; Giménez, Luis E.

    2009-01-01

    Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M2-muscarinic acetylcholine receptors (M2AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M2AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M2AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [3H]-N-methyl scopolamine ([3H]-NMS) in allosterism binding assays. A peptide corresponding to the M2AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [3H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [3H]-NMS dissociation right shifted from an IC50 of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 × 10−8, 1.33 × 10−7, and 2.0 × 10−7 mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M2AChRs as a positive cooperativity effect on acetylcholine action. PMID:18702010

  3. Novel AChE Inhibitors for Sustainable Insecticide Resistance Management

    PubMed Central

    Alout, Haoues; Labbé, Pierrick; Berthomieu, Arnaud; Djogbénou, Luc; Leonetti, Jean-Paul; Fort, Philippe; Weill, Mylène

    2012-01-01

    Resistance to insecticides has become a critical issue in pest management and it is particularly chronic in the control of human disease vectors. The gravity of this situation is being exacerbated since there has not been a new insecticide class produced for over twenty years. Reasoned strategies have been developed to limit resistance spread but have proven difficult to implement in the field. Here we propose a new conceptual strategy based on inhibitors that preferentially target mosquitoes already resistant to a currently used insecticide. Application of such inhibitors in rotation with the insecticide against which resistance has been selected initially is expected to restore vector control efficacy and reduce the odds of neo-resistance. We validated this strategy by screening for inhibitors of the G119S mutated acetylcholinesterase-1 (AChE1), which mediates insensitivity to the widely used organophosphates (OP) and carbamates (CX) insecticides. PyrimidineTrione Furan-substituted (PTF) compounds came out as best hits, acting biochemically as reversible and competitive inhibitors of mosquito AChE1 and preferentially inhibiting the mutated form, insensitive to OP and CX. PTF application in bioassays preferentially killed OP-resistant Culex pipiens and Anopheles gambiae larvae as a consequence of AChE1 inhibition. Modeling the evolution of frequencies of wild type and OP-insensitive AChE1 alleles in PTF-treated populations using the selectivity parameters estimated from bioassays predicts a rapid rise in the wild type allele frequency. This study identifies the first compound class that preferentially targets OP-resistant mosquitoes, thus restoring OP-susceptibility, which validates a new prospect of sustainable insecticide resistance management. PMID:23056599

  4. Aches and pains during pregnancy

    MedlinePlus

    ... medlineplus.gov/ency/patientinstructions/000580.htm Aches and pains during pregnancy To use the sharing features on ... the end of your pregnancy, tell your provider. Pain in Your Lower Abdomen (Belly) or Groin Most ...

  5. Blocking M2 muscarinic receptor signaling inhibits tumor growth and reverses epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC)

    PubMed Central

    Zhao, Qingnan; Gu, Xiajing; Zhang, Chun; Lu, Qin; Chen, Hongzhuan; Xu, Lu

    2015-01-01

    Lung cancers express non-neuronal, cholinergic autoparacrine loop, which facilitates tumor growth. Interruption of M3 muscarinic cholinergic signaling has been reported to inhibit small cell lung cancer (SCLC) growth. The purpose of this study is to investigate if blocking autoparacrine muscarinic cholinergic signaling could inhibit non-small cell lung cancer (NSCLC) growth and possible underlying mechanisms. Our results showed that PC9 and A549 cells expressed all 5 subtypes of muscarinic receptor (mAChR) and blocking M2 mAChR (M2R) signaling using selective antagonist methoctramine or short hairpin RNA (shRNA) inhibited tumor cell proliferation in vitro and in vivo. Consistent with AChR agonists stimulating p44/42 MAPK (Erk1/2) and Akt phosphorylation, blocking M2R signaling decreased MAPK and Akt phosphorylation, indicating that non-neuronal ACh functions as an autoparacrine growth factor signaling in part through activation of M2R and downstream MAPK and Akt pathways. Importantly, further studies revealed that blocking M2R signaling also reversed epithelial-mesenchymal transition (EMT) in vitro and in vivo, indicating that non-neuronal ACh promotes EMT partially through activation of M2R. These findings demonstrate that M2R plays a role in the growth and progression of NSCLC and suggest M2R antagonists may be an efficacious adjuvant therapy for NSCLC. PMID:25778781

  6. A Comparison of MyoD1 and Fetal Acetylcholine Receptor Expression in Childhood Tumors and Normal Tissues

    PubMed Central

    Gattenloehner, Stefan; Dockhorn-Dworniczak, Barbara; Leuschner, Ivo; Vincent, Angela; Müller-Hermelink, Hans-Konrad; Marx, Alexander

    1999-01-01

    Detection of minimal residual disease or micrometastases in rhabdomyosarcoma (RMS) has been an unresolved problem in 70 to 80% of RMS patients. In patients with alveolar type RMS, which harbors chromosomal translocations and produces tumor-specific fusion products, polymerase chain reaction (PCR)-based diagnosis is clear-cut. In the more frequent embryonal RMS, however, no such PCR-based marker has been described. Recently it has been suggested that the PCR-based detection of MyoD1 may be a valuable adjunct in the diagnosis of minimal disease in embryonal RMS. We report here that MyoD1 mRNA is not specific for RMS, but can be amplified from ex vivo samples of many other childhood tumors and some normal tissues. By contrast, simultaneous amplification of α and γ subunit message of the fetal type acetylcholine receptor (AChR), by a novel duplex PCR, and the quantification of both transcripts resulting in a α/γAChR ratio <1 was 100% sensitive in alveolar (n = 8) and embryonal (n = 10) RMS. Moreover, γAChR was not detected in other childhood (n = 27) or adult tumors (n = 12), or normal tissues, except thymus. The high sensitivity and specificity of the method were confirmed by the successful detection of five cases of cytologically or molecularly verified RMS bone marrow micrometastases among 47 bone marrow samples from childhood tumor patients. By contrast, MyoD1 showed no amplification because of its low level of transcription. We conclude that mRNA of the fetal type AChR is a more specific and (about 100 times) more sensitive marker for the molecular detection of RMS than MyoD1, and thus appears to be a promising candidate for the detection of minimal disease in RMS lacking tumor-specific translocations. PMID:11272905

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

    PubMed

    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

  8. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

    PubMed

    Mine, N; Taniguchi, W; Nishio, N; Izumi, N; Miyazaki, N; Yamada, H; Nakatsuka, T; Yoshida, M

    2015-04-01

    Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective α4β2 nicotinic receptor antagonist, dihydro-β-erythroidine hydrobromide (DhβE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective α7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that α4β2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-α4β2 and non-α7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic transmission in the spinal ventral horn. PMID:25613686

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

    PubMed

    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

  10. Anti-acetylcholine receptor antibodies.

    PubMed Central

    Vincent, A; Newsom Davis, J

    1980-01-01

    Early suggestions that a humoral factor might be implicated in the disorder of neuromuscular transmission in myasthenia gravis have been confirmed by the detection of anti-AChR antibody in 85-90% of the patients with generalised disease and in 75% of cases with restricted ocular myasthenia. Plasma exchange reveals that serum anti-AChR usually has an inverse relationship to muscle strength and present evidence indicates that patients responding to thymectomy and immunosuppressive durg treatment usually show a consistent decline in serum anti-AChR titres. The antibody is heterogeneous and can lead to a loss of muscle AChR by several mechanisms. Anti-AChR is produced in the thymus in relatively small amounts. Anti-AChR antibody synthesis by thymic lymphocytes and pokeweed stimulated peripheral lymphocytes in culture provides a means of studying the effect of different lymphocyte populations in vitro. Analysis of clinical, immunological and HLA antigen characteristics in MG suggest that more than one mechanism may underlie the breakdown in tolerance to AChR, leading to the production of anti-AChR antibodies. PMID:7400823

  11. In vitro proliferative responses and antibody titers specific to human acetylcholine receptor synthetic peptides in patients with myasthenia gravis and relation to HLA class II genes.

    PubMed Central

    Brocke, S; Brautbar, C; Steinman, L; Abramsky, O; Rothbard, J; Neumann, D; Fuchs, S; Mozes, E

    1988-01-01

    To investigate which parts of the acetylcholine receptor are involved in the initiation and development of myasthenia gravis (MG), peptides representing different sequences of the human acetylcholine receptor alpha-subunit were synthesized. These peptides were tested for their ability to stimulate T cells of myasthenic patients and healthy control patients in proliferation assays and to bind to sera antibodies. Three of eight peptides discriminated significantly between the two groups in the proliferation assay, as well as in their ability to bind to serum antibodies. HLA-DR3 and DR5 were associated with proliferative responses to specific AChR peptides in the group of myasthenics. Acetylcholine receptor epitopes that might play a specific role in myasthenia gravis thus were demonstrated. PMID:2461962

  12. A comparison of MyoD1 and fetal acetylcholine receptor expression in childhood tumors and normal tissues: implications for the molecular diagnosis of minimal disease in rhabdomyosarcomas.

    PubMed

    Gattenloehner, S; Dockhorn-Dworniczak, B; Leuschner, I; Vincent, A; Müller-Hermelink, H K; Marx, A

    1999-11-01

    Detection of minimal residual disease or micrometastases in rhabdomyosarcoma (RMS) has been an unresolved problem in 70 to 80% of RMS patients. In patients with alveolar type RMS, which harbors chromosomal translocations and produces tumor-specific fusion products, polymerase chain reaction (PCR)-based diagnosis is clear-cut. In the more frequent embryonal RMS, however, no such PCR-based marker has been described. Recently it has been suggested that the PCR-based detection of MyoD1 may be a valuable adjunct in the diagnosis of minimal disease in embryonal RMS. We report here that MyoD1 mRNA is not specific for RMS, but can be amplified from ex vivo samples of many other childhood tumors and some normal tissues. By contrast, simultaneous amplification of alpha and gamma subunit message of the fetal type acetylcholine receptor (AChR), by a novel duplex PCR, and the quantification of both transcripts resulting in a alpha/gammaAChR ratio <1 was 100% sensitive in alveolar (n = 8) and embryonal (n = 10) RMS. Moreover, gammaAChR was not detected in other childhood (n = 27) or adult tumors (n = 12), or normal tissues, except thymus. The high sensitivity and specificity of the method were confirmed by the successful detection of five cases of cytologically or molecularly verified RMS bone marrow micrometastases among 47 bone marrow samples from childhood tumor patients. By contrast, MyoD1 showed no amplification because of its low level of transcription. We conclude that mRNA of the fetal type AChR is a more specific and (about 100 times) more sensitive marker for the molecular detection of RMS than MyoD1, and thus appears to be a promising candidate for the detection of minimal disease in RMS lacking tumor-specific translocations. PMID:11272905

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

    PubMed

    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

  14. Sorbs1 and -2 Interact with CrkL and Are Required for Acetylcholine Receptor Cluster Formation

    PubMed Central

    Hallock, Peter T.; Chin, Sherry; Blais, Steven; Neubert, Thomas A.

    2015-01-01

    Crk and CrkL are noncatalytic adaptor proteins necessary for the formation of neuromuscular synapses which function downstream of muscle-specific kinase (MuSK), a receptor tyrosine kinase expressed in skeletal muscle, and the MuSK binding protein Dok-7. How Crk/CrkL regulate neuromuscular endplate formation is not known. To better understand the roles of Crk/CrkL, we identified CrkL binding proteins using mass spectrometry and have identified Sorbs1 and Sorbs2 as two functionally redundant proteins that associate with the initiating MuSK/Dok-7/Crk/CrkL complex, regulate acetylcholine receptor (AChR) clustering in vitro, and are localized at synapses in vivo. PMID:26527617

  15. A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

    PubMed

    Fuchs, P A; Murrow, B W

    1992-04-22

    The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle. PMID:1355909

  16. Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor

    SciTech Connect

    Dowding, A.J.; Hall, Z.W.

    1987-10-06

    The authors have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of ..cap alpha..-bungarotoxin (..cap alpha..-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block ..cap alpha..-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit ..cap alpha..-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds ..cap alpha..-BuTx with a forward rate constant of 0.98 x 10/sup 5/ M/sup -1/ s/sup -1/, d-tubocurarine (dTC) with a K/sub D/ of (6.8 +/- 0.3) x 10/sup -8/ M, and pancuronium with a K/sub D/ of (1.9 +/- 1.0) x 10/sup -9/ M. The site recognized by B mAbs binds ..cap alpha..-BuTx with a forward rate constant of 9.3 x 10/sup 5/ M/sup -1/ s/sup -1/, dTC with a K/sub D/ of (4.6 +/- 0.3) x 10/sup -6/ M, and pancurionium with a K/sub D/ of (9.3 +/- 0.8) x 10/sup -6/ M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by ..cap alpha..-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.

  17. Synthesis and biological activities of indolizine derivatives as alpha-7 nAChR agonists.

    PubMed

    Xue, Yu; Tang, Jingshu; Ma, Xiaozhuo; Li, Qing; Xie, Bingxue; Hao, Yuchen; Jin, Hongwei; Wang, Kewei; Zhang, Guisen; Zhang, Liangren; Zhang, Lihe

    2016-06-10

    Human α7 nicotinic acetylcholine receptor (nAChR) is a promising therapeutic target for the treatment of schizophrenia accompanied with cognitive impairment. Herein, we report the synthesis and agonistic activities of a series of indolizine derivatives targeting to α7 nAChR. The results show that all synthesized compounds have affinity to α7 nAChR and some give strong agonistic activity, particularly most active agonists show higher potency than control EVP-6124. The docking and structure-activity relationship studies provide insights to develop more potent novel α7 nAChR agonists. PMID:26994846

  18. Kinetic evidence that desensitized nAChR may promote transitions of active nAChR to desensitized states during sustained exposure to agonists in skeletal muscle.

    PubMed

    Manthey, Arthur A

    2006-06-01

    During prolonged exposure of postjunctional nicotinic acetylcholine receptors (nAChR) of skeletal muscle to acetylcholine (ACh), agonist-activated nAChR (nAChRa) gradually fall into a refractory "desensitized" state (nAChRd), which no longer supports the high-conductance channel openings characteristic of the initially active nAChRa. In the present study, the possibility was examined that nAChRd, rather than simply constituting a passive "trap" for nAChRa, may actively promote further conversions of nAChRa to nAChRd in a formally autocatalytic manner. Single-ion whole-cell voltage-clamp currents (Na+ and Li+ in separate trials) were measured using two KCl-filled capillary electrodes (5-10 MOmega) implanted at the postjunctional locus of single frog skeletal muscle fibers (Rana pipiens) equilibrated in 30 mM K+ bath media to eliminate mechanical responses. Various nAChR agonists (carbamylcholine, acetylcholine, suberyldicholine) at different concentrations were delivered focally by positive pressure microjet. It was found that the decline of postmaximal agonist-induced currents under these different conditions (driven by the growth of the subpool of nAChRd) consistently followed an autocatalytic logistic rule modified for population growth of fixed units in a planar array: [Formula: see text] (where y represents the remaining agonist-induced current at time t, A=initial maximum current, and n is a constant). Some further experimental features that might result from a self-promoting growth of nAChRd were also tested, namely, (1) the effect of increased nAChRa and (2) the effect of increased nAChRd. Increase in agonist concentration of the superfusate, by increasing the planar density of active nAChRa at the outset, should enhance the probability of autocatalytic interactions with emerging nAChRd, hence, the rate of decline of agonist-induced current, and this was a consistent finding under all conditions tested. Raising the initial level of desensitized nAChRd by

  19. Readthrough acetylcholinesterase (AChE-R) and regulated necrosis: pharmacological targets for the regulation of ovarian functions?

    PubMed

    Blohberger, J; Kunz, L; Einwang, D; Berg, U; Berg, D; Ojeda, S R; Dissen, G A; Fröhlich, T; Arnold, G J; Soreq, H; Lara, H; Mayerhofer, A

    2015-01-01

    Proliferation, differentiation and death of ovarian cells ensure orderly functioning of the female gonad during the reproductive phase, which ultimately ends with menopause in women. These processes are regulated by several mechanisms, including local signaling via neurotransmitters. Previous studies showed that ovarian non-neuronal endocrine cells produce acetylcholine (ACh), which likely acts as a trophic factor within the ovarian follicle and the corpus luteum via muscarinic ACh receptors. How its actions are restricted was unknown. We identified enzymatically active acetylcholinesterase (AChE) in human ovarian follicular fluid as a product of human granulosa cells. AChE breaks down ACh and thereby attenuates its trophic functions. Blockage of AChE by huperzine A increased the trophic actions as seen in granulosa cells studies. Among ovarian AChE variants, the readthrough isoform AChE-R was identified, which has further, non-enzymatic roles. AChE-R was found in follicular fluid, granulosa and theca cells, as well as luteal cells, implying that such functions occur in vivo. A synthetic AChE-R peptide (ARP) was used to explore such actions and induced in primary, cultured human granulosa cells a caspase-independent form of cell death with a distinct balloon-like morphology and the release of lactate dehydrogenase. The RIPK1 inhibitor necrostatin-1 and the MLKL-blocker necrosulfonamide significantly reduced this form of cell death. Thus a novel non-enzymatic function of AChE-R is to stimulate RIPK1/MLKL-dependent regulated necrosis (necroptosis). The latter complements a cholinergic system in the ovary, which determines life and death of ovarian cells. Necroptosis likely occurs in the primate ovary, as granulosa and luteal cells were immunopositive for phospho-MLKL, and hence necroptosis may contribute to follicular atresia and luteolysis. The results suggest that interference with the enzymatic activities of AChE and/or interference with necroptosis may be novel

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

    PubMed Central

    Abraham, Nikita; Paul, Blessy; Ragnarsson, Lotten; Lewis, Richard J.

    2016-01-01

    Nicotinic acetylcholine receptors (nAChR) are ligand gated ion channels, identified as therapeutic targets for a range of human diseases. Drug design for nAChR related disorders is increasingly using structure-based approaches. Many of these structural insights for therapeutic lead development have been obtained from co-crystal structures of nAChR agonists and antagonists with the acetylcholine binding protein (AChBP). AChBP is a water soluble, structural and functional homolog of the extracellular, ligand-binding domain of nAChRs. Currently, AChBPs are recombinantly expressed in eukaryotic expression systems for structural and biophysical studies. Here, we report the establishment of an Escherichia coli (E. coli) expression system that significantly reduces the cost and time of production compared to the existing expression systems. E. coli can efficiently express unglycosylated AChBP for crystallography and makes the expression of isotopically labelled forms feasible for NMR. We used a pHUE vector containing an N-terminal His-tagged ubiquitin fusion protein to facilitate AChBP expression in the soluble fractions, and thus avoid the need to recover protein from inclusion bodies. The purified protein yield obtained from the E. coli expression system is comparable to that obtained from existing AChBP expression systems. E. coli expressed AChBP bound nAChR agonists and antagonists with affinities matching those previously reported. Thus, the E. coli expression system significantly simplifies the expression and purification of functional AChBP for structural and biophysical studies. PMID:27304486

  1. Menthol Alone Upregulates Midbrain nAChRs, Alters nAChR Subtype Stoichiometry, Alters Dopamine Neuron Firing Frequency, and Prevents Nicotine Reward.

    PubMed

    Henderson, Brandon J; Wall, Teagan R; Henley, Beverley M; Kim, Charlene H; Nichols, Weston A; Moaddel, Ruin; Xiao, Cheng; Lester, Henry A

    2016-03-01

    Upregulation of β2 subunit-containing (β2*) nicotinic acetylcholine receptors (nAChRs) is implicated in several aspects of nicotine addiction, and menthol cigarette smokers tend to upregulate β2* nAChRs more than nonmenthol cigarette smokers. We investigated the effect of long-term menthol alone on midbrain neurons containing nAChRs. In midbrain dopaminergic (DA) neurons from mice containing fluorescent nAChR subunits, menthol alone increased the number of α4 and α6 nAChR subunits, but this upregulation did not occur in midbrain GABAergic neurons. Thus, chronic menthol produces a cell-type-selective upregulation of α4* nAChRs, complementing that of chronic nicotine alone, which upregulates α4 subunit-containing (α4*) nAChRs in GABAergic but not DA neurons. In mouse brain slices and cultured midbrain neurons, menthol reduced DA neuron firing frequency and altered DA neuron excitability following nAChR activation. Furthermore, menthol exposure before nicotine abolished nicotine reward-related behavior in mice. In neuroblastoma cells transfected with fluorescent nAChR subunits, exposure to 500 nm menthol alone also increased nAChR number and favored the formation of (α4)3(β2)2 nAChRs; this contrasts with the action of nicotine itself, which favors (α4)2(β2)3 nAChRs. Menthol alone also increases the number of α6β2 receptors that exclude the β3 subunit. Thus, menthol stabilizes lower-sensitivity α4* and α6 subunit-containing nAChRs, possibly by acting as a chemical chaperone. The abolition of nicotine reward-related behavior may be mediated through menthol's ability to stabilize lower-sensitivity nAChRs and alter DA neuron excitability. We conclude that menthol is more than a tobacco flavorant: administered alone chronically, it alters midbrain DA neurons of the nicotine reward-related pathway. PMID:26961950

  2. Synaptic abnormalities of mice lacking toll-like receptor (TLR)-9.

    PubMed

    Patel, V; Patel, A M; McArdle, J J

    2016-06-01

    Motor, sensory, and autonomic abnormalities are reported for toll-like receptor 9 (TLR9) knock-out (KO) mice. However, a physiological role of TLR9 in the nervous system is largely unknown. Since altered synaptic transmission can contribute to sensory and motor abnormalities, we evaluated neuromuscular junction (NMJ) function and morphology of TLR9 KO mice. Triangularis sterni nerve-muscle preparations were dissected from TLR9 KO and age-matched control mice. Two-electrode voltage clamp of the motor endplate revealed that the amplitude and frequency of miniature end plate currents (mEPCs) for TLR9 KO NMJs were significantly greater than control. In contrast, mean endplate current (EPC, 1Hz) amplitude was equivalent to control. The ratio of mean EPC to mean mEPC amplitude indicated a decline of quantal content (m) for TLR9 KO NMJs. Furthermore, m declined more rapidly than control in response to 50-Hz stimulus trains. A rightward shift of the mEPC amplitude distribution suggested formation of vesicles containing larger amounts of acetylcholine (ACh). Staining with rhodamine α-bungarotoxin revealed a significant decline of endplate size in TLR9 KO mice. This alteration may result from ACh-induced decline of acetylcholine receptor (AChR) expression resulting from increased frequency and amplitude of mEPCs. At the same time, excessive spontaneous vesicular ACh release may initiate retrograde suppression of excitation-secretion coupling. These data suggest a novel role of TLR9 in the development of the NMJ. PMID:26955780

  3. Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes.

    PubMed Central

    Montal, M; Labarca, P; Fredkin, D R; Suarez-Isla, B A

    1984-01-01

    The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing. PMID:6324900

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

    PubMed Central

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

    2015-01-01

    Acetylcholine receptors are pentameric ligand–gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

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

    PubMed

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

    2015-12-01

    Acetylcholine receptors are pentameric ligand-gated channels involved in excitatory neuro-transmission in both vertebrates and invertebrates. In nematodes, they represent major targets for cholinergic agonist or antagonist anthelmintic drugs. Despite the large diversity of acetylcholine-receptor subunit genes present in nematodes, only a few receptor subtypes have been characterized so far. Interestingly, parasitic nematodes affecting human or animal health possess two closely related members of this gene family, acr-26 and acr-27 that are essentially absent in free-living or plant parasitic species. Using the pathogenic parasitic nematode of ruminants, Haemonchus contortus, as a model, we found that Hco-ACR-26 and Hco-ACR-27 are co-expressed in body muscle cells. We demonstrated that co-expression of Hco-ACR-26 and Hco-ACR-27 in Xenopus laevis oocytes led to the functional expression of an acetylcholine-receptor highly sensitive to the anthelmintics morantel and pyrantel. Importantly we also reported that ACR-26 and ACR-27, from the distantly related parasitic nematode of horses, Parascaris equorum, also formed a functional acetylcholine-receptor highly sensitive to these two drugs. In Caenorhabditis elegans, a free-living model nematode, we demonstrated that heterologous expression of the H. contortus and P. equorum receptors drastically increased its sensitivity to morantel and pyrantel, mirroring the pharmacological properties observed in Xenopus oocytes. Our results are the first to describe significant molecular determinants of a novel class of nematode body wall muscle AChR. PMID:26625142

  6. From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR

    PubMed Central

    Lin, Bo; Xu, Manyu; Zhu, Xiaopeng; Wu, Yong; Liu, Xi; Zhangsun, Dongting; Hu, Yuanyan; Xiang, Shi-Hua; Kasheverov, Igor E.; Tsetlin, Victor I.; Wang, Xinquan; Luo, Sulan

    2016-01-01

    Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes. PMID:26925840

  7. From crystal structure of α-conotoxin GIC in complex with Ac-AChBP to molecular determinants of its high selectivity for α3β2 nAChR.

    PubMed

    Lin, Bo; Xu, Manyu; Zhu, Xiaopeng; Wu, Yong; Liu, Xi; Zhangsun, Dongting; Hu, Yuanyan; Xiang, Shi-Hua; Kasheverov, Igor E; Tsetlin, Victor I; Wang, Xinquan; Luo, Sulan

    2016-01-01

    Acetylcholine binding proteins (AChBPs) are unique spatial homologs of the ligand-binding domains of nicotinic acetylcholine receptors (nAChRs), and they reproduce some pharmacological properties of nAChRs. X-ray crystal structures of AСhBP in complex with α-conotoxins provide important insights into the interactions of α-conotoxins with distinct nAChR subtypes. Although considerable efforts have been made to understand why α-conotoxin GIC is strongly selective for α3β2 nAChR, this question has not yet been solved. Here we present the structure of α-conotoxin GIC in complex with Aplysia californica AChBP (Ac-AChBP) at a resolution of 2.1 Å. Based on this co-crystal structure complemented with molecular docking data, we suggest the key residues of GIC in determining its high affinity and selectivity for human α3β2 vs α3β4 nAChRs. These suggestions were checked by radioligand and electrophysiology experiments, which confirmed the functional role of detected contacts for GIC interactions with Ac-AChBP and α3β2 nAChR subtypes. While GIC elements responsible for its high affinity binding with Ac-AChBP and α3β2 nAChR were identified, our study also showed the limitations of computer modelling in extending the data from the X-ray structures of the AChBP complexes to all nAChR subtypes. PMID:26925840

  8. Circannual rhythms of acetylcholinesterase (AChE) activity in the freshwater fish Cnesterodon decemmaculatus.

    PubMed

    Menéndez-Helman, Renata J; Ferreyroa, Gisele V; dos Santos Afonso, Maria; Salibián, Alfredo

    2015-01-01

    The use of biomarkers as a tool to assess responses of organisms exposed to pollutants in toxicity bioassays, as well as in aquatic environmental risk assessment protocols, requires the understanding of the natural fluctuation of the particular biomarker. The aim of this study was to characterize the intrinsic variations of acetylcholinesterase (AChE) activity in tissues of a native freshwater teleost fish to be used as biomarker in toxicity tests, taking into account both seasonal influence and fish size. Specific AChE activity was measured by the method of Ellman et al. (1961) in homogenates of fish anterior section finding a seasonal variability. The highest activity was observed in summer, decreasing significantly below 40% in winter. The annual AChE activity cycle in the anterior section was fitted to a sinusoidal function with a period of 11.2 months. Moreover, an inverse relationship between enzymatic activity and the animal size was established. The results showed that both the fish length and seasonal variability affect AChE activity. AChE activity in fish posterior section showed a similar trend to that in the anterior section, while seasonal variations of the activity in midsection were observed but differences were not statistically significant. In addition, no relationship between AChE and total tissue protein was established in the anterior and posterior sections suggesting that the circannual rhythms observed are AChE-specific responses. Results highlight the importance of considering both the fish size and season variations to reach valid conclusions when AChE activity is employed as neurotoxicity biomarker. PMID:25450939

  9. Effect of pharmaceuticals exposure on acetylcholinesterase (AchE) activity and on the expression of AchE gene in the monogonont rotifer, Brachionus koreanus.

    PubMed

    Rhee, Jae-Sung; Kim, Bo-Mi; Jeong, Chang-Bum; Park, Heum Gi; Leung, Kenneth Mei Yee; Lee, Young-Mi; Lee, Jae-Seong

    2013-11-01

    Pharmaceuticals are widely used in human and veterinary medicine. However, they are emerging as a significant contaminant in aquatic environments through wastewater. Due to the persistent and accumulated properties of pharmaceuticals via the food web, their potential harmful effects on aquatic animals are a great concern. In this study, we investigated the effects of six pharmaceuticals: acetaminophen, ATP; atenolol, ATN; carbamazepine, CBZ; oxytetracycline, OTC; sulfamethoxazole, SMX; and trimethoprim, TMP on acetylcholinesterase (AChE; EC 3.1.1.7) activity and its transcript expression with chlorpyrifos (as a positive control) in the monogonont rotifer, Brachionus koreanus. ATP, CBZ, and TMP exposure also remarkably inhibited Bk-AChE activity at 100 μg/L (24 h) and 1000 μg/L (12 h and 24 h). ATP, CBZ, and TMP exposure showed a significant decrease in the Bk-AChE mRNA level in a concentration-dependent manner. However, in the case of OTC and SMX, a slight decrease in Bk-AChE mRNA expression was found but only at the highest concentration. The time-course experiments showed that ATP positively induced Bk-AChE mRNA 12 h after exposure at both 100 and 1000 μg/L, while the Bk-AChE mRNA expression was significantly downregulated over 6 to 24 h after exposure to 1000 μg/L of CBZ, OTC, SMX, and TMP. Our findings suggest that Bk-AChE would be a useful biomarker for risk assessment of pharmaceutical compounds as an early signal of their toxicity in aquatic environments. Particularly, ATP, CBZ, and TMP may have a toxic cholinergic effect on rotifer B. koreanus by inhibiting AChE activity. PMID:24028855

  10. The gating isomerization of neuromuscular acetylcholine receptors

    PubMed Central

    Auerbach, Anthony

    2010-01-01

    Acetylcholine receptor-channels are allosteric proteins that isomerize (‘gate’) between conformations that have a low vs. high affinity for the transmitter and conductance for ions. In order to comprehend the mechanism by which the affinity and conductance changes are linked it is of value to know the magnitude, timing and distribution of energy flowing through the system. Knowing both the di- and unliganded gating equilibrium constants (E2 and E0) is a foundation for understanding the AChR gating mechanism and for engineering both the ligand and the protein to operate in predictable ways. In adult mouse neuromuscular receptors activated by acetylcholine, E2= 28 and E0≈ 6.5 × 10−7. At each (equivalent) transmitter binding site acetylcholine provides ∼5.2 kcal mol−1 to motivate the isomerization. The partial agonist choline provides ∼3.3 kcal mol−1. The relative time of a residue's gating energy change is revealed by the slope of its rate–equilibrium constant relationship. A map of this parameter suggests that energy propagates as a conformational cascade between the transmitter binding sites and the gate region. Although gating energy changes are widespread throughout the protein, some residues are particularly sensitive to perturbations. Several specific proposals for the structural events that comprise the gating conformational cascade are discussed. PMID:19933754

  11. Agonist-induced changes in the structure of the acetylcholine receptor M2 regions revealed by photoincorporation of an uncharged nicotinic noncompetitive antagonist.

    PubMed

    White, B H; Cohen, J B

    1992-08-01

    To characterize structural changes induced in the nicotinic acetylcholine receptor (AChR) by agonists, we have mapped the sites of photoincorporation of the cholinergic noncompetitive antagonist 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (]125I]TID) in the presence and absence of 50 microM carbamylcholine. [125I]TID binds to the AChR with similar affinity under both these conditions, but agonist inhibits photoincorporation into all subunits by greater than 75% (White, B. H., Howard, S., Cohen, S. G., and Cohen, J. B. (1991) J. Biol. Chem. 266, 21595-21607). [125I]TID-labeled sites on the beta- and delta-subunits were identified by amino-terminal sequencing of both cyanogen bromide (CNBr) and tryptic fragments purified by Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reversed-phase high-performance liquid chromatography. In the absence of agonist, [125I]TID specifically labels homologous aliphatic residues (beta L-257, delta L-265, beta V-261, and delta V-269) in the M2 region of both subunits. In the presence of agonist, labeling of these residues is reduced approximately 90%, and the distribution of labeled residues is broadened to include a homologous set of serine residues at the amino terminus of M2. In the beta-subunit residues beta S-250, beta S-254, beta L-257, and beta V-261 are all labeled in the presence of carbamylcholine. This pattern of labeling supports an alpha-helical model for M2 with the labeled face forming the ion channel lumen. The observed redistribution of label in the resting and desensitized states provides the first direct evidence for an agonist-dependent rearrangement of the M2 helices. The efficient labeling of the resting state channel in a region capable of structural change also suggests a plausible model for AChR gating in which the aliphatic residues labeled by [125I]TID form a permeability barrier to the passage of ions. We also report increased labeling of the M1 region of the delta

  12. The α2-subunit of the nicotinic cholinergic receptor is specifically expressed in medial subpallium-derived cells of mammalian amygdala.

    PubMed

    Pombero, Ana; Martinez, Salvador

    2015-08-01

    Nicotinic acetylcholine receptor (nAChR) subtypes are expressed in specific neuronal populations, which are involved in numerous neural functions such as sleep, fatigue, anxiety, and cognition, as well as the central processing of pain and food intake. Moreover, mutations in nAChRs subunits have been related to frontal lobe epilepsy, neurodegenerative diseases, and other neurological disorders, including schizophrenia and attention deficit and hyperactivity disorder (ADHD). Previous studies have shown that the α2-subunit of the AChR (Chrna2) is expressed in the basal forebrain, in the septum, and in some amygdalar nuclei in the adult rodent brain. However, although the importance of this amygdalar expression in emotion-related behavior and the physiopathology of neuropsychiatric disorders has been accepted, a detailed study of the Chrna2 expression pattern during development has been lacking. In this study we found that Chrna2 is specifically expressed in medial subpallium-derived amygdalar nuclei from early developmental stages to adult. This finding could help us to better understand the role of Chrna2 in the differentiation and functional maturation of amygdalar neurons involved in cholinergic-regulated emotional behavior. PMID:25641263

  13. Natural AChE Inhibitors from Plants and their Contribution to Alzheimer’s Disease Therapy

    PubMed Central

    Murray, Ana Paula; Faraoni, María Belén; Castro, María Julia; Alza, Natalia Paola; Cavallaro, Valeria

    2013-01-01

    As acetylcholinesterase (AChE) inhibitors are an important therapeutic strategy in Alzheimer’s disease, efforts are being made in search of new molecules with anti-AChE activity. The fact that naturally-occurring compounds from plants are considered to be a potential source of new inhibitors has led to the discovery of an important number of secondary metabolites and plant extracts with the ability of inhibiting the enzyme AChE, which, according to the cholinergic hypothesis, increases the levels of the neurotransmitter acetylcholine in the brain, thus improving cholinergic functions in patients with Alzheimer’s disease and alleviating the symptoms of this neurological disorder. This review summarizes a total of 128 studies which correspond to the most relevant research work published during 2006-2012 (1st semester) on plant-derived compounds, plant extracts and essential oils found to elicit AChE inhibition. PMID:24381530

  14. Beyond acetylcholinesterase inhibitors for treating Alzheimer's disease: α7-nAChR agonists in human clinical trials.

    PubMed

    Russo, Patrizia; Del Bufalo, Alessandra; Frustaci, Alessandra; Fini, Massimo; Cesario, Alfredo

    2014-01-01

    The neuronal nicotinic alpha7-acetylcholine receptor (α7-nAChR) is a promising and attractive drug target for improving cognitive deficits in neuropsychiatric and neurological disorders such as Alzheimer's disease (AD). α7-nAChR belongs to the family of ligand gated ion channels. α7-nAChR is expressed in key brain regions (e.g. pre- and frontal cortex, hippocampus). It is involved in essential cognitive functions such as memory, thinking, comprehension, learning capacity, calculation, orientation, language, and judgment. α7-nAChR binds to amyloid peptide (Aβ) inducing either receptor activation or inhibition in an Aβ concentration-dependent mode. Aβ oligomers induce τ phosphorylation via α7-nAChR activation. α7-nAChR agonists and/or α7-nAChR positive allosteric modulators may be useful in AD therapy. The current review enlightens: (i) α7-nAChR neurobiology, (ii) α7-nAChR role in cognition and (iii) in AD, and (iv) the clinical status of the most promising molecules for the treatment of cognitive dysfunction in AD. PMID:24641224

  15. Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera

    PubMed Central

    Bren, Nina; Cheng, Kevin; Gomoto, Ryan; Chen, Lin; Sine, Steven M.

    2014-01-01

    To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Å2 (1 Å = 0.1 nm) of surface area, within which Arg36 and Phe32 from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr184 from loop-C of α7, while Asp30 of α-btx forms a hydrogen bond with the hydroxy group of Tyr184. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr184 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings high-light structural principles by which α-neurotoxins interact with nicotinic receptors. PMID:23800261

  16. Neuroprotective effects of donepezil against Aβ42-induced neuronal toxicity are mediated through not only enhancing PP2A activity but also regulating GSK-3β and nAChRs activity.

    PubMed

    Noh, Min-Young; Koh, Seong H; Kim, Sung-Min; Maurice, Tangui; Ku, Sae-Kwang; Kim, Seung H

    2013-11-01

    The main purpose of this study was to evaluate whether donepezil, acetylcholinesterase inhibitor, shown to play a protective role through inhibiting glycogen synthesis kinase-3β (GSK-3β) activity, could also exert neuroprotective effects by stimulating protein phosphatase 2A (PP2A) activity in the amyloid-beta (Aβ)42-induced neuronal toxicity model of Alzheimer's disease. In Aβ42-induced toxic conditions, each PP2A and GSK-3β activity measured at different times showed time-dependent reverse pattern toward the direction of accelerating neuronal deaths with the passage of time. In addition, donepezil pre-treatment showed dose-dependent stepwise increase of neuronal viability and stimulation of PP2A activity. However, such effects on them were significantly reduced through the depletion of PP2A activity with either okadaic acid or PP2Ac siRNA. In spite of blocked PP2A activity in this Aβ42 insult, however, donepezil pretreatment showed additional significant recovering effect on neuronal viability when compared to the value without donepezil. Moreover, donepezil partially recovered its dephosphorylating effect on hyperphosphorylated tau induced by Aβ42. This observation led us to assume that additional mechanisms of donepezil, including its inhibitory effect on GSK-3β activity and/or the activation role of nicotinic acetylcholine receptors (nAChRs), might be involved. Taken together, our results suggest that the neuroprotective effects of donepezil against Aβ42-induced neurotoxicity are mediated through activation of PP2A, but its additional mechanisms including regulation of GSK-3β and nAChRs activity would partially contribute to its effects. We investigated neuroprotective mechanisms of donepezil against Aβ42 toxicity: Donepezil increased neuronal viability with reduced p-tau by enhancing PP2A activity. Despite of blocked PP2A activity, donepezil showed additional recovering effect on neuronal viability, which findings led us to assume that additional

  17. Functional expression and axonal transport of α7 nAChRs by peptidergic nociceptors of rat dorsal root ganglion.

    PubMed

    Shelukhina, Irina; Paddenberg, Renate; Kummer, Wolfgang; Tsetlin, Victor

    2015-07-01

    In recent pain studies on animal models, α7 nicotinic acetylcholine receptor (nAChR) agonists demonstrated analgesic, anti-hyperalgesic and anti-inflammatory effects, apparently acting through some peripheral receptors. Assuming possible involvement of α7 nAChRs on nociceptive sensory neurons, we investigated the morphological and neurochemical features of the α7 nAChR-expressing subpopulation of dorsal root ganglion (DRG) neurons and their ability to transport α7 nAChR axonally. In addition, α7 receptor activity and its putative role in pain signal neurotransmitter release were studied. Medium-sized α7 nAChR-expressing neurons prevailed, although the range covered all cell sizes. These cells accounted for one-fifth of total medium and large DRG neurons and <5% of small ones. 83.2% of α7 nAChR-expressing DRG neurons were peptidergic nociceptors (CGRP-immunopositive), one half of which had non-myelinated C-fibers and the other half had myelinated Aδ- and likely Aα/β-fibers, whereas 15.2% were non-peptidergic C-fiber nociceptors binding isolectin B4. All non-peptidergic and a third of peptidergic α7 nAChR-bearing nociceptors expressed TRPV1, a capsaicin-sensitive noxious stimulus transducer. Nerve crush experiments demonstrated that CGRPergic DRG nociceptors axonally transported α7 nAChRs both to the spinal cord and periphery. α7 nAChRs in DRG neurons were functional as their specific agonist PNU282987 evoked calcium rise enhanced by α7-selective positive allosteric modulator PNU120596. However, α7 nAChRs do not modulate neurotransmitter CGRP and glutamate release from DRG neurons since nicotinic ligands affected neither their basal nor provoked levels, showing the necessity of further studies to elucidate the true role of α7 nAChRs in those neurons. PMID:24706047

  18. Atomic interactions of neonicotinoid agonists with AChBP: Molecular recognition of the distinctive electronegative pharmacophore

    SciTech Connect

    Talley, Todd T.; Harel, Michal; Hibbs, Ryan E.; Radi, Zoran; Tomizawa, Motohiro; Casida, John E.; Taylor, Palmer

    2008-07-28

    Acetylcholine-binding proteins (AChBPs) from mollusks are suitable structural and functional surrogates of the nicotinic acetylcholine receptors when combined with transmembrane spans of the nicotinic receptor. These proteins assemble as a pentamer with identical ACh binding sites at the subunit interfaces and show ligand specificities resembling those of the nicotinic receptor for agonists and antagonists. A subset of ligands, termed the neonicotinoids, exhibit specificity for insect nicotinic receptors and selective toxicity as insecticides. AChBPs are of neither mammalian nor insect origin and exhibit a distinctive pattern of selectivity for the neonicotinoid ligands. We define here the binding orientation and determinants of differential molecular recognition for the neonicotinoids and classical nicotinoids by estimates of kinetic and equilibrium binding parameters and crystallographic analysis. Neonicotinoid complex formation is rapid and accompanied by quenching of the AChBP tryptophan fluorescence. Comparisons of the neonicotinoids imidacloprid and thiacloprid in the binding site from Aplysia californica AChBP at 2.48 and 1.94 {angstrom} in resolution reveal a single conformation of the bound ligands with four of the five sites occupied in the pentameric crystal structure. The neonicotinoid electronegative pharmacophore is nestled in an inverted direction compared with the nicotinoid cationic functionality at the subunit interfacial binding pocket. Characteristic of several agonists, loop C largely envelops the ligand, positioning aromatic side chains to interact optimally with conjugated and hydrophobic regions of the neonicotinoid. This template defines the association of interacting amino acids and their energetic contributions to the distinctive interactions of neonicotinoids.

  19. Lymphocyte-derived ACh regulates local innate but not adaptive immunity

    PubMed Central

    Reardon, Colin; Duncan, Gordon S.; Brüstle, Anne; Brenner, Dirk; Tusche, Michael W.; Olofsson, Peder S.; Rosas-Ballina, Mauricio; Tracey, Kevin J.; Mak, Tak W.

    2013-01-01

    Appropriate control of immune responses is a critical determinant of health. Here, we show that choline acetyltransferase (ChAT) is expressed and ACh is produced by B cells and other immune cells that have an impact on innate immunity. ChAT expression occurs in mucosal-associated lymph tissue, subsequent to microbial colonization, and is reduced by antibiotic treatment. MyD88-dependent Toll-like receptor up-regulates ChAT in a transient manner. Unlike the previously described CD4+ T-cell population that is stimulated by norepinephrine to release ACh, ChAT+ B cells release ACh after stimulation with sulfated cholecystokinin but not norepinephrine. ACh-producing B-cells reduce peritoneal neutrophil recruitment during sterile endotoxemia independent of the vagus nerve, without affecting innate immune cell activation. Endothelial cells treated with ACh in vitro reduced endothelial cell adhesion molecule expression in a muscarinic receptor-dependent manner. Despite this ability, ChAT+ B cells were unable to suppress effector T-cell function in vivo. Therefore, ACh produced by lymphocytes has specific functions, with ChAT+ B cells controlling the local recruitment of neutrophils. PMID:23297238

  20. Avarol derivatives as competitive AChE inhibitors, non hepatotoxic and neuroprotective agents for Alzheimer's disease.

    PubMed

    Tommonaro, Giuseppina; García-Font, Nuria; Vitale, Rosa Maria; Pejin, Boris; Iodice, Carmine; Cañadas, Sixta; Marco-Contelles, José; Oset-Gasque, María Jesús

    2016-10-21

    Avarol is a marine sesquiterpenoid hydroquinone, previously isolated from the marine sponge Dysidea avara Schmidt (Dictyoceratida), with antiinflammatory, antitumor, antioxidant, antiplatelet, anti-HIV, and antipsoriatic effects. Recent findings indicate that some thio-avarol derivatives exhibit acetylcholinesterase (AChE) inhibitory activity. The multiple pharmacological properties of avarol, thio-avarol and/or their derivatives prompted us to continue the in vitro screening, focusing on their AChE inhibitory and neuroprotective effects. Due to the complex nature of Alzheimer's disease (AD), there is a renewed search for new, non hepatotoxic anticholinesterasic compounds. This paper describes the synthesis and in vitro biological evaluation of avarol-3'-thiosalicylate (TAVA) and thiosalycil-prenyl-hydroquinones (TPHs), as non hepatotoxic anticholinesterasic agents, showing a good neuroprotective effect on the decreased viability of SHSY5Y human neuroblastoma cells induced by oligomycin A/rotenone and okadaic acid. A molecular modeling study was also undertaken on the most promising molecules within the series to elucidate their AChE binding modes and in particular the role played by the carboxylate group in enzyme inhibition. Among them, TPH4, bearing a geranylgeraniol substituent, is the most significant Electrophorus electricus AChE (EeAChE) inhibitor (IC50 = 6.77 ± 0.24 μM), also endowed with a moderate serum horse butyrylcholinesterase (eqBuChE) inhibitory activity, being also the least hepatotoxic and the best neuroprotective compound of the series. Thus, TPHs represents a new family of synthetic compounds, chemically related to the natural compound avarol, which has been discovered for the potential treatment of AD. Findings prove the relevance of TPHs as a new possible generation of competitive AChE inhibitors pointing out the importance of the salycilic substituents on the hydroquinone ring. Since these compounds do not belong to the class of

  1. Age-related hearing loss: GABA, nicotinic acetylcholine and NMDA receptor expression changes in spiral ganglion neurons of the mouse.

    PubMed

    Tang, X; Zhu, X; Ding, B; Walton, J P; Frisina, R D; Su, J

    2014-02-14

    Age-related hearing loss - presbycusis - is the number one communication disorder and most prevalent neurodegenerative condition of our aged population. Although speech understanding in background noise is quite difficult for those with presbycusis, there are currently no biomedical treatments to prevent, delay or reverse this condition. A better understanding of the cochlear mechanisms underlying presbycusis will help lead to future treatments. Objectives of the present study were to investigate GABAA receptor subunit α1, nicotinic acetylcholine (nACh) receptor subunit β2, and N-methyl-d-aspartate (NMDA) receptor subunit NR1 mRNA and protein expression changes in spiral ganglion neurons (SGN) of the CBA/CaJ mouse cochlea, that occur in age-related hearing loss, utilizing quantitative immunohistochemistry and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) techniques. We found that auditory brainstem response (ABR) thresholds shifted over 40dB from 3 to 48kHz in old mice compared to young adults. DPOAE thresholds also shifted over 40dB from 6 to 49kHz in old mice, and their amplitudes were significantly decreased or absent in the same frequency range. SGN density decreased with age in basal, middle and apical turns, and SGN density of the basal turn declined the most. A positive correlation was observed between SGN density and ABR wave 1amplitude. mRNA and protein expression of GABAAR α1 and AChR β2 decreased with age in SGNs in the old mouse cochlea. mRNA and protein expression of NMDAR NR1 increased with age in SGNs of the old mice. These findings demonstrate that there are functionally-relevant age-related changes of GABAAR, nAChR, NMDAR expression in CBA mouse SGNs reflecting their degeneration, which may be related to functional changes in cochlear synaptic transmission with age, suggesting biological mechanisms for peripheral age-related hearing loss. PMID:24316061

  2. Flexibility versus “rigidity” of the functional architecture of AChE active center

    PubMed Central

    Shafferman, Avigdor; Barak, Dov; Stein, Dana; Kronman, Chanoch; Velan, Baruch; Greig, Nigel H.; Ordentlich, Arie

    2008-01-01

    Functional architecture of the AChE active center appears to be characterized by both structural “rigidity”, necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the “rigid” environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the “hydrophobic patch” which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the “hydrophobic patch” in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the

  3. Molecular docking of fisetin with AD associated AChE, ABAD and BACE1 proteins

    PubMed Central

    Dash, Raju; Emran, Talha Bin; Uddin, Mir Muhammad Nasir; Islam, Ashekul; Junaid, Md

    2014-01-01

    Alzheimer׳s disease (AD) is one of the most common dementias showing slow progressive cognitive decline. Progression of intracerebral accumulation of beta amyloid (Aβ) peptides by the action of amyloid binding alcohol dehydrogenase (ABAD), a mitochondrial enzyme and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the degradation of Acetylcholinesterase (AChE) the main pathological characteristics of AD. Therefore, it is of interest to evaluate the importance of fisetin (a flavonol that belongs to the flavonoid group of polyphenols) binding with AChE, ABAD and BACE1 proteins. Docking experiment of fisetin with these proteins using two different tools namely iGEMDOCK and FlexX show significant binding with acceptable binding values. Thus, the potential inhibitory role of fisetin with AD associated proteins is documented. PMID:25352723

  4. Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: ( sup 3 H)nicotine as an agonist photoaffinity label

    SciTech Connect

    Middleton, R.E.; Cohen, J.B. )

    1991-07-16

    The agonist ({sup 3}H)nicotine was used as a photoaffinity label for the acetylcholine binding sties on the Torpedo nicotinic acetylcholine receptor (AChR). ({sup 3}H)Nicotine binds at equilibrium with K{sub eq} = 0.6 {mu}M to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with ({sup 3}H)nicotine resulted in covalent incorporation into the {alpha}- and {gamma}-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the {alpha}-subunit was labeled via both agonist sites but the {gamma}-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Chymotryptic digestion of the {alpha}-subunit confirmed that Try-198 was the principal amino acid labeled by ({sup 3}H)nicotine. This confirmation required a novel radiosequencing strategy employing o-phthalaldehyde ({sup 3}H)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.

  5. Cotinine Exposure Increases Fallopian Tube PROKR1 Expression via Nicotinic AChRα-7

    PubMed Central

    Shaw, Julie L.V.; Oliver, Elizabeth; Lee, Kai-Fai; Entrican, Gary; Jabbour, Henry N.; Critchley, Hilary O.D.; Horne, Andrew W.

    2010-01-01

    Tubal ectopic pregnancy (EP) is the most common cause of maternal mortality in the first trimester of pregnancy; however, its etiology is uncertain. In EP, embryo retention within the Fallopian tube (FT) is thought to be due to impaired smooth muscle contractility (SMC) and alterations in the tubal microenvironment. Smoking is a major risk factor for EP. FTs from women with EP exhibit altered prokineticin receptor-1 (PROKR1) expression, the receptor for prokineticins (PROK). PROK1 is angiogenic, regulates SMC, and is involved in intrauterine implantation. We hypothesized that smoking predisposes women to EP by altering tubal PROKR1 expression. Sera/FT were collected at hysterectomy (n = 21). Serum levels of the smoking metabolite, cotinine, were measured by enzyme-linked immunosorbent assay. FTs were analyzed by q-RT-PCR, immunohistochemistry, and Western blotting for expression of PROKR1 and the predicted cotinine receptor, nicotinic acetylcholine receptor α-7 (AChRα−7). FT explants (n = 4) and oviductal epithelial cells (cell line OE-E6/E7) were treated with cotinine and an nAChRα−7 antagonist. PROKR1 transcription was higher in FTs from smokers (P < 0.01). nAChRα−7 expression was demonstrated in FT epithelium. Cotinine treatment of FT explants and OE-E6/E7 cells increased PROKR1 expression (P < 0.05), which was negated by cotreatment with nAChRα−7 antagonist. Smoking targets human FTs via nAChRα−7 to increase tubal PROKR1, leading to alterations in the tubal microenvironment that could predispose to EP. PMID:20864676

  6. Tribendimidine: Mode of Action and nAChR Subtype Selectivity in Ascaris and Oesophagostomum

    PubMed Central

    Robertson, Alan P.; Puttachary, Sreekanth; Buxton, Samuel K.; Martin, Richard J.

    2015-01-01

    The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype

  7. Amine substitution of quinazolinones leads to selective nanomolar AChE inhibitors with 'inverted' binding mode.

    PubMed

    Darras, Fouad H; Wehle, Sarah; Huang, Guozheng; Sotriffer, Christoph A; Decker, Michael

    2014-09-01

    Selective and nanomolar acetylcholinesterase inhibitors were obtained by connecting tri- and tetracyclic quinazolinones-previously described as moderately active and unselective cholinesterase (ChE) inhibitors-via a hydroxyl group in para position to an anilinic nitrogen with different amines linked via a three carbon atom spacer. These tri- and tetracyclic quinazolinones containing different alicyclic ring sizes and connected to tertiary amines were docked to a high-resolution hAChE crystal structure to investigate the preferred binding mode in relation to results obtained by experimental structure-activity relationships. While the 'classical orientation' locating the heterocycle in the active site was rarely found, an alternative binding mode with the basic aliphatic amine in the active center ('inverted' orientation) was obtained for most compounds. Analyses of extended SARs based on this inverted binding mode are able to explain the compounds' binding affinities at AChE. PMID:25047936

  8. Baculovirus expression, biochemical characterization and organophosphate sensitivity of rBmAChE1, rBmAChE2, and rBmAChE3 of Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhipicephalus (Boophilus) microplus cDNAs, BmAChE1, BmAChE2, and BmAChE3,were previously identified as presumptively encoding acetylcholinesterases, but biochemical identity was confirmed only for recombinant BmAChE3. In the present study, four recombinant BmAChE1 constructs and single recombinant c...

  9. Modulatory effects of α7 nAChRs on the immune system and its relevance for CNS disorders.

    PubMed

    Kalkman, Hans O; Feuerbach, Dominik

    2016-07-01

    The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer's disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer's disease, Parkinson's disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders. PMID:26979166

  10. Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon.

    PubMed

    Herkert, Nadja M; Aurbek, Nadine; Eyer, Peter; Thiermann, Horst; Worek, Franz

    2010-05-01

    Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models. PMID:20156534

  11. Nicotinic Acetylcholine Receptors in Sensory Cortex

    ERIC Educational Resources Information Center

    Metherate, Raju

    2004-01-01

    Acetylcholine release in sensory neocortex contributes to higher-order sensory function, in part by activating nicotinic acetylcholine receptors (nAChRs). Molecular studies have revealed a bewildering array of nAChR subtypes and cellular actions; however, there is some consensus emerging about the major nAChR subtypes and their functions in…

  12. Anti-inflammatory role of microglial alpha7 nAChRs and its role in neuroprotection.

    PubMed

    Egea, Javier; Buendia, Izaskun; Parada, Esther; Navarro, Elisa; León, Rafael; Lopez, Manuela G

    2015-10-15

    Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the central nervous system, being expressed in neurons and non-neuronal cells, where they participate in a variety of physiological responses like memory, learning, locomotion, attention, among others. We will focus on the α7 nAChR subtype, which has been implicated in neuroprotection, synaptic plasticity and neuronal survival, and is considered as a potential therapeutic target for several neurological diseases. Oxidative stress and neuroinflammation are currently considered as two of the most important pathological mechanisms common in neurodegenerative diseases such as Alzheimer, Parkinson or Huntington diseases. In this review, we will first analysed the distribution and expression of nAChR in mammalian brain. Then, we focused on the function of the α7 nAChR subtype in neuronal and non-neuronal cells and its role in immune responses (cholinergic anti-inflammatory pathway). Finally, we will revise the anti-inflammatory pathway promoted via α7 nAChR activation that is related to recruitment and activation of Jak2/STAT3 pathway, which on the one hand inhibits NF-κB nuclear translocation, and on the other hand, activates the master regulator of oxidative stress Nrf2/HO-1. This review provides a profound insight into the role of the α7 nAChR subtype in microglia and point out to microglial α7/HO-1 pathway as an anti-inflammatory therapeutic target. PMID:26232730

  13. Going up in Smoke? A Review of nAChRs-based Treatment Strategies for Improving Cognition in Schizophrenia

    PubMed Central

    Boggs, Douglas L.; Carlson, Jon; Cortes-Briones, Jose; Krystal, John H.; D’Souza, D. Cyril

    2015-01-01

    Cognitive impairment is known to be a core deficit in schizophrenia. Existing treatments for schizophrenia have limited efficacy against cognitive impairment. The ubiquitous use of nicotine in this population is thought to reflect an attempt by patients to self-medicate certain symptoms associated with the illness. Concurrently there is evidence that nicotinic receptors that have lower affinity for nicotine are more important in cognition. Therefore, a number of medications that target nicotinic acetylcholine receptors (nAChRs) have been tested or are in development. In this article we summarize the clinical evidence of nAChRs dysfunction in schizophrenia and review clinical studies testing either nicotine or nicotinic medications for the treatment of cognitive impairment in schizophrenia. Some evidence suggests beneficial effects of nAChRs based treatments for the attentional deficits associated with schizophrenia. Standardized cognitive test batteries have failed to capture consistent improvements from drugs acting at nAChRs. However, more proximal measures of brain function, such as ERPs relevant to information processing impairments in schizophrenia, have shown some benefit. Further work is necessary to conclude that nAChRs based treatments are of clinical utility in the treatment of cognitive deficits of schizophrenia. PMID:24345265

  14. Crystal structure of a human neuronal nAChR extracellular domain in pentameric assembly: Ligand-bound α2 homopentamer.

    PubMed

    Kouvatsos, Nikolaos; Giastas, Petros; Chroni-Tzartou, Dafni; Poulopoulou, Cornelia; Tzartos, Socrates J

    2016-08-23

    In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2β2 nAChR and validated the functional importance of specific residues in α2 and β2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases. PMID:27493220

  15. Effect of calcium on nicotine-induced current expressed by an atypical alpha-bungarotoxin-insensitive nAChR2.

    PubMed

    Thany, Steeve H; Courjaret, Raphael; Lapied, Bruno

    2008-06-27

    Two distinct native alpha-bungarotoxin (alpha-Bgt)-insensitive nicotinic acetylcholine receptors (nAChRs), named nAChR1 and nAChR2, were identified in the cockroach Periplaneta americana dorsal unpaired median (DUM) neurons. They differed in their electrophysiological, pharmacological properties and intracellular regulation pathways. nAChR2 being an atypical nicotinic receptor closed upon agonist application and its current-voltage relationship resulted from a reduction in potassium conductance. In this study, using whole-cell patch-clamp technique, we demonstrated that calcium modulated nAChR2-mediated nicotine response. Under 0.5 microM alpha-Bgt and 20 mM d-tubocurarine, the nicotine-induced inward current amplitude was strongly reduced in the presence of intracellularly applied BAPTA or bath application of calcium-free solution. In addition, using cadmium chloride, we showed that nicotine response was modulated by extracellular calcium through plasma membrane calcium channels. Moreover, extracellular application of caffeine and thapsigargin reduced nAChR2-mediated response. Together these experiments revealed a complex calcium-dependent regulation of nAChR2. PMID:18485593

  16. Cross-talk between α7 nAchR and NMDAR revealed by protein profiling.

    PubMed

    Zhang, Hailong; Li, Tao; Li, Shupeng; Liu, Fang

    2016-01-10

    Functional regulation of NMDA receptor (NMDAR) by the activation of α7 nicotinic acetylcholine receptor (α7nAChR) has been reported, although the molecular signaling pathway underlying this process remains largely unknown. We employed a label-free quantitative proteomics approach to identify potential intracellular molecules and pathways that might be involved in the functional cross-talk between NMDAR and α7nAChR. 43 proteins showed significantly expression changes after choline treatment in which 35 out of 43 proteins was significantly altered by co-treatment with NMDA. Western blot analysis verified proteins expression determined by LC-MS. Furthermore, protein expression in vivo in neurons from fetal rats were visualized and quantified by Confocal microscopy,which showed consistency of relative changes of AHA-1 expressionmeasured by LC-MS and Western blot. Biological network analysis of differently expressed proteins found 21 kind of biological pathways involved. Of those pathways, 6 pathways were directly involved in regulation of neurotransmitters. Lastly, the levels of neurotransmitters (dopamine, glutamate, GABA and 5-HT) were measured by HPLC-ECD. Co-treatment choline/NMDA significantly enhances the release of dopamine, glutamate and GABA and dramatically decrease 5-HT to only 65% of control level, which is consist with this protein interaction network analysis, providing an additional evidence for the cross-talk between NMDAR and α7nAChR. PMID:26498070

  17. Continuous flow immobilized enzyme reactor-tandem mass spectrometry for screening of AChE inhibitors in complex mixtures.

    PubMed

    Forsberg, Erica M; Green, James R A; Brennan, John D

    2011-07-01

    A method is described for identifying bioactive compounds in complex mixtures based on the use of capillary-scale monolithic enzyme-reactor columns for rapid screening of enzyme activity. A two-channel nanoLC system was used to continuously infuse substrate coupled with automated injections of substrate/small molecule mixtures, optionally containing the chromogenic Ellman reagent, through sol-gel derived acetylcholinesterase (AChE) doped monolithic columns. This is the first report of AChE encapsulated in monolithic silica for use as an immobilized enzyme reactor (IMER), and the first use of such IMERs for mixture screening. AChE IMER columns were optimized to allow rapid functional screening of compound mixtures based on changes in the product absorbance or the ratio of mass spectrometric peaks for product and substrate ions in the eluent. The assay had robust performance and produced a Z' factor of 0.77 in the presence of 2% (v/v) DMSO. A series of 52 mixtures consisting of 1040 compounds from the Canadian Compound Collection of bioactives was screened and two known inhibitors, physostigmine and 9-aminoacridine, were identified from active mixtures by manual deconvolution. The activity of the compounds was confirmed using the enzyme reactor format, which allowed determination of both IC(50) and K(I) values. Screening results were found to correlate well with a recently published fluorescence-based microarray screening assay for AChE inhibitors. PMID:21591743

  18. Mechanisms of Barbiturate Inhibition of Acetylcholine Receptor Channels

    PubMed Central

    Dilger, James P.; Boguslavsky, Rebecca; Barann, Martin; Katz, Tamir; Vidal, Ana Maria

    1997-01-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts is 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 μM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 μM, n = 1.09; for barbital, Ki = 1900 μM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. <0.1 ms at the Ki). Pentobarbital binds ≥10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at >100 μM pentobarbital. PMID:9089445

  19. Mechanisms of barbiturate inhibition of acetylcholine receptor channels.

    PubMed

    Dilger, J P; Boguslavsky, R; Barann, M; Katz, T; Vidal, A M

    1997-03-01

    We used patch clamp techniques to study the inhibitory effects of pentobarbital and barbital on nicotinic acetylcholine receptor channels from BC3H-1 cells. Single channel recording from outside-out patches reveals that both drugs cause acetylcholine-activated channel events to occur in bursts. The mean duration of gaps within bursts in 2 ms for 0.1 mM pentobarbital and 0.05 ms for 1 mM barbital. In addition, 1 mM barbital reduces the apparent single channel current by 15%. Both barbiturates decrease the duration of openings within a burst but have only a small effect on the burst duration. Macroscopic currents were activated by rapid perfusion of 300 microM acetylcholine to outside-out patches. The concentration dependence of peak current inhibition was fit with a Hill function; for pentobarbital, Ki = 32 microM, n = 1.09; for barbital, Ki = 1900 microM, n = 1.24. Inhibition is voltage independent. The kinetics of inhibition by pentobarbital are at least 30 times faster than inhibition by barbital (3 ms vs. < 0.1 ms at the Ki). Pentobarbital binds > or = 10-fold more tightly to open channels than to closed channels; we could not determine whether the binding of barbital is state dependent. Experiments performed with both barbiturates reveal that they do not compete for a single binding site on the acetylcholine receptor channel protein, but the binding of one barbiturate destabilizes the binding of the other. These results support a kinetic model in which barbiturates bind to both open and closed states of the AChR and block the flow of ions through the channel. An additional, lower-affinity binding site for pentobarbital may explain the effects seen at > 100 microM pentobarbital. PMID:9089445

  20. The postsynaptic submembrane machinery at the neuromuscular junction: requirement for rapsyn and the utrophin/dystrophin-associated complex.

    PubMed

    Banks, Glen B; Fuhrer, Christian; Adams, Marvin E; Froehner, Stanley C

    2003-01-01

    Neuromuscular synapse formation is brought about by a complex bi-directional exchange of information between the innervating motor neuron and its target skeletal muscle fiber. Agrin, a heparin sulfate proteoglycan, is released from the motor nerve terminal to activate its muscle-specific kinase (MuSK) receptor that leads to a second messenger cascade requiring rapsyn to ultimately bring about AChR clustering in the muscle membrane. Rapsyn performs many functions in skeletal muscle. First, rapsyn and AChRs co-target to the postsynatic apparatus. Second, rapsyn may self associate to stabilize and promote AChR clustering. Third, rapsyn is essential for AChR cluster formation. Fourth, rapsyn is required to transduce the agrin-evoked MuSK phosphorylation signal to AChRs. Finally, rapsyn links AChRs to the utrophin-associated complex, which appears to be required for AChR stabilization as well as maturation of the neuromuscular junction. Proteins within the utrophin-associated complex such as alpha-dystrobrevin and alpha-syntrophin are also important for signaling events that affect neuromuscular synapse stability and function. Here we review our current understanding of the role of the postsynaptic-submembrane machinery involving rapsyn and the utrophin-associated complex at the neuromuscular synapse. In addition we briefly review how these studies of the neuromuscular junction relate to GABAergic and glycinergic synapses in the CNS. PMID:15034263

  1. Acetylcholinesterases of Rhipicephalus (Boophilus) microplus – Multiple gene expression presents an opportune model system for elucidation of multiple functions of AChEs.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Acetylcholinesterase (AChE) is a key neural enzyme of both vertebrates and invertebrates, and is the biochemical target of organophosphate and carbamate pesticides for invertebrates, as well as vertebrate nerve agents, e.g., soman, tabun, VX, and others. AChE inhibitors are also key drugs among thos...

  2. Synthesis and comparison of the biological activity of monocyclic phosphonate, difluorophosphonate and phosphate analogs of the natural AChE inhibitor cyclophostin.

    PubMed

    Martin, Benjamin P; Vasilieva, Elena; Dupureur, Cynthia M; Spilling, Christopher D

    2015-12-15

    New monocyclic phosphate, phosphonate and difluorophosphonate analogs of the natural AChE inhibitor cyclophostin were synthesized and their activity toward human AChE examined. Surprisingly, the phosphate, phosphonate, and difluorophosphonate analogs all showed diminished activity when compared with the natural product. PMID:26585276

  3. Functionality and stability data of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

    PubMed

    Padilla-Morales, Luis F; Colón-Sáez, José O; González-Nieves, Joel E; Quesada-González, Orestes; Lasalde-Dominicci, José A

    2016-03-01

    The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR) rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16). The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique. PMID:26870753

  4. Antinociceptive effect of spirocyclopiperazinium salt compound LXM-15 via activating peripheral α7 nAChR and M4 mAChR in mice.

    PubMed

    Zhao, Xia; Ye, Jia; Sun, Qi; Xiong, Yulan; Li, Runtao; Jiang, Yimin

    2011-01-01

    The aim of this study was to evaluate the antinociceptive effects and potential mechanisms of the spirocyclopiperazinium compound LXM-15. We found that LXM-15 produced significant antinociceptive effects in a dose- and time-dependent manner in mice. The maximum inhibition ratio was 70% in the acetic acid writhing test; the effect started at 1.0 h, peaked at 2.0 h with the MPEs of 61%, and persisted 3.5 h in the hot-plate test; LXM-15 reduced the time spent licking or biting the injected paw remarkably with inhibitions of 53% in formalin test. LXM-15 did not affect motor coordination, spontaneous activity, body temperature, heart rate, or liver enzyme activity, the LD(50) values was 616.26 μmol/kg. The antinociceptive effect of LXM-15 was blocked by mecamylamine, hexamethonium, atropine or atropine methylnitrate, and was also blocked by MLA, tropicamide. In contrast, the effect was not blocked by naloxone. Meanwhile, competition receptor binding assays showed LXM-15 can bind to α7 nAChR or M4 mAChR. Our studies show that LXM-15 may be via activating peripheral α7 nicotnic and M4 muscarinic receptors, resulted in antinociceptive effects. PMID:21035471

  5. Gripped by Gout: Avoiding the Ache and Agony

    MedlinePlus

    ... please review our exit disclaimer . Subscribe Gripped by Gout Avoiding the Ache and Agony Sudden, painful swelling ... toe is often the first warning sign of gout. It can affect other joints as well. Without ...

  6. Enantiopure Cyclopropane-Bearing Pyridyldiazabicyclo[3.3.0]octanes as Selective α4β2-nAChR Ligands.

    PubMed

    Onajole, Oluseye K; Eaton, J Brek; Lukas, Ronald J; Brunner, Dani; Thiede, Lucinda; Caldarone, Barbara J; Kozikowski, Alan P

    2014-11-13

    We report the synthesis and characterization of a series of enantiopure 5-cyclopropane-bearing pyridyldiazabicyclo[3.3.0]octanes that display low nanomolar binding affinities and act as functional agonists at α4β2-nicotinic acetylcholine receptor (nAChR) subtype. Structure-activity relationship studies revealed that incorporation of a cyclopropane-containing side chain at the 5-position of the pyridine ring provides ligands with improved subtype selectivity for nAChR β2 subunit-containing nAChR subtypes (β2*-nAChRs) over β4*-nAChRs compared to the parent compound 4. Compound 15 exhibited subnanomolar binding affinity for α4β2- and α4β2*-nAChRs with negligible interaction. Functional assays confirm selectivity for α4β2-nAChRs. Furthermore, using the SmartCube assay system, this ligand showed antidepressant, anxiolytic, and antipsychotic features, while mouse forced-swim assay further confirm the antidepressant-like property of 15. PMID:25408831

  7. Acetylcholinesterase (AChE) is an important link in the apoptotic pathway induced by hyperglycemia in Y79 retinoblastoma cell line.

    PubMed

    Masha'our, R Shehadeh; Heinrich, R; Garzozi, H J; Perlman, I

    2012-01-01

    Acetylcholinesterase (AChE) expression was found to be induced in the mammalian CNS, including the retina, by different types of stress leading to cellular apoptosis. Here, we tested possible involvement of AChE in hyperglycemia-induced apoptosis in a retinal cell line. Y79 retinoblastoma cells were incubated in starvation media (1% FBS and 1 mg/ml glucose) for 16-24 h, and then exposed to hyperglycemic environment by raising extracellular glucose concentrations to a final level of 3.5 mg/ml or 6 mg/ml. Similar levels of mannitol were used as control for hyperosmolarity. Cells were harvested at different time intervals for analysis of apoptosis and AChE protein expression. Apoptosis was detected by the cleavage of Poly ADP-ribose polymerase (PARP) using western blot, and by Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) assay. AChE protein expression and activity was detected by western blot and by the Karnovsky and Roots method, respectively. Mission(TM) shRNA for AChE was used to inhibit AChE protein expression. Treating Y79 cells with 3.5 mg/ml of glucose, but not with 3.5 mg/ml mannitol, induced apoptosis which was confirmed by TUNEL assay and by cleavage of PARP. A part of the signaling pathway accompanying the apoptotic process involved up-regulation of the AChE-R variant and an N-extended AChE variant as verified at the mRNA and protein level. Inhibition of AChE protein expression by shRNA protected Y79 cell from entering the apoptotic pathway. Our data suggest that expression of an N-extended AChE variant, most probably an R isoform, is involved in the apoptotic pathway caused by hyperglycemia in Y79 cells. PMID:22685426

  8. The Role of nAChR and Calcium Signaling in Pancreatic Cancer Initiation and Progression

    PubMed Central

    Schaal, Courtney; Padmanabhan, Jaya; Chellappan, Srikumar

    2015-01-01

    Pancreatic cancer shows a strong correlation with smoking and the current therapeutic strategies have been relatively ineffective in improving the survival of patients. Efforts have been made over the past many years to understand the molecular events that drive the initiation and progression of pancreatic cancer, especially in the context of smoking. It has become clear that components of tobacco smoke not only initiate these cancers, especially pancreatic ductal adenocarcinomas (PDACs) through their mutagenic properties, but can also promote the growth and metastasis of these tumors by stimulating cell proliferation, angiogenesis, invasion and epithelial-mesenchymal transition. Studies in cell culture systems, animal models and human samples have shown that nicotinic acetylcholine receptor (nAChR) activation enhances these tumor-promoting events by channeling signaling through multiple pathways. In this context, signaling through calcium channels appear to facilitate pancreatic cancer growth by itself or downstream of nAChRs. This review article highlights the role of nAChR downstream signaling events and calcium signaling in the growth, metastasis as well as drug resistance of pancreatic cancer. PMID:26264026

  9. Evidence for a role for α6(∗) nAChRs in l-dopa-induced dyskinesias using Parkinsonian α6(∗) nAChR gain-of-function mice.

    PubMed

    Bordia, T; McGregor, M; McIntosh, J M; Drenan, R M; Quik, M

    2015-06-01

    l-Dopa-induced dyskinesias (LIDs) are a serious side effect of dopamine replacement therapy for Parkinson's disease. The mechanisms that underlie LIDs are currently unclear. However, preclinical studies indicate that nicotinic acetylcholine receptors (nAChRs) play a role, suggesting that drugs targeting these receptors may be of therapeutic benefit. To further understand the involvement of α6β2(∗) nAChRs in LIDs, we used gain-of-function α6(∗) nAChR (α6L9S) mice that exhibit a 20-fold enhanced sensitivity to nAChR agonists. Wildtype (WT) and α6L9S mice were lesioned by unilateral injection of 6-hydroxydopamine (6-OHDA, 3μg/ml) into the medial forebrain bundle. Three to 4wk later, they were administered l-dopa (3mg/kg) plus benserazide (15mg/kg) until stably dyskinetic. l-dopa-induced abnormal involuntary movements (AIMs) were similar in α6L9S and WT mice. WT mice were then given nicotine in the drinking water in gradually increasing doses to a final 300μg/ml, which resulted in a 40% decline AIMs. By contrast, there was no decrease in AIMs in α6L9S mice at a maximally tolerated nicotine dose of 20μg/ml. However, the nAChR antagonist mecamylamine (1mg/kg ip 30min before l-dopa) reduced l-dopa-induced AIMs in both α6L9S and WT mice. Thus, both a nAChR agonist and antagonist decreased AIMs in WT mice, but only the antagonist was effective in α6L9S mice. Since nicotine appears to reduce LIDs via desensitization, hypersensitive α6β2(∗) nAChRs may desensitize less readily. The present data show that α6β2(∗) nAChRs are key regulators of LIDs, and may be useful therapeutic targets for their management in Parkinson's disease. PMID:25813704

  10. Potent AChE enzyme inhibition activity of Zizyphus oxyphylla: A new source of antioxidant compounds.

    PubMed

    Mazhar, Farhana; Khanum, Raisa; Ajaib, Muhammad; Jahangir, Muhammad

    2015-11-01

    The purpose of this study was to assess the antioxidant potential and enzyme inhibition of various fractions of Zizyphus oxyphylla. The plant metabolites were extracted in methanol and partitioned with n-hexane, chloroform, ethyl acetate and n-butanol successively. Phytochemical screening showed presence of alkaloids, terpenoids and flavonoids in ethyl acetate and n-butanol fractions. The antioxidant potential and acetylcholine esterase assay of all these fractions and remaining aqueous fraction was evaluated by using reported methods. The results revealed that chloroform soluble fraction exhibited highest percent inhibition of DPPH radical as compared to other fractions. It showed 95.01 ± 0.37% inhibition of DPPH radical at a concentration of 120 μg/mL. The IC₅₀ of this fraction was 13.20 ± 0.27 μg/mL, relative to butylated hydroxytoluene (BHT, a reference standard), having IC₅₀ of 12.10 ± 0.29 μg/mL. It also showed highest total antioxidant activity i.e. 1.723 ± 0.34 as well as highest FRAP value (339.5 ± 0.57 TE μm/mL) and highest total phenolic contents (142.65 ± 1.20 GAE mg/g) as compared to the other studied fractions. The fractions were also studied for Acetylcholine esterase enzyme (AChE) enzyme inhibition activity and n-butanol soluble fraction exhibited maximum inhibition (95.5 ± 0.13 mg/mL with IC50 =9.58 ± 0.08 mg/mL relative to galanthamine (13.26 ± 0.73 mg/mL), while n- hexane soluble fraction (165.15 ± 0.94 mg/mL) showed non-significant. We are still working to isolate pure compounds for active fractions targeting potent inhibition responsible for some activities. PMID:26639499

  11. Evaluation of the Toxicity, AChE Activity and DNA Damage Caused by Imidacloprid on Earthworms, Eisenia fetida.

    PubMed

    Wang, Kai; Qi, Suzhen; Mu, Xiyan; Chai, Tingting; Yang, Yang; Wang, Dandan; Li, Dongzhi; Che, Wunan; Wang, Chengju

    2015-10-01

    Imidacloprid is a well-known pesticide and it is timely to evaluate its toxicity to earthworms (Eisenia fetida). In the present study, the effect of imidacloprid on reproduction, growth, acetylcholinesterase (AChE) and DNA damage in earthworms was assessed using an artificial soil medium. The median lethal concentration (LC50) and the median number of hatched cocoons (EC50) of imidacloprid to earthworms was 3.05 and 0.92 mg/kg respectively, the lowest observed effect concentration of imidacloprid about hatchability, growth, AChE activity and DNA damage was 0.02, 0.5, 0.1 and 0.5 mg/kg, respectively. PMID:26293707

  12. Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

    PubMed

    Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

    2014-01-01

    The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively

  13. Toxicological and Biochemical Characterizations of AChE in Phosalone-Susceptible and Resistant Populations of the Common Pistachio Psyllid, Agonoscena pistaciae

    PubMed Central

    Alizadeh, Ali; Talebi-Jahromi, Khalil; Hosseininaveh, Vahid; Ghadamyari, Mohammad

    2014-01-01

    The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (KM) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively

  14. Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs

    PubMed Central

    Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R.; McIntosh, J. Michael; Brunzell, Darlene H.; Cannon, Jason R.; Drenan, Ryan M.

    2014-01-01

    α6β2* nAChRs in the ventral tegmental area (VTA) to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9’S mice) that are hypersensitive to nicotine and endogenous acetylcholine (ACh). Evoked extracellular dopamine (DA) levels were enhanced in α6L9’S NAc slices compared to control, non-transgenic (nonTg) slices. Extracellular DA levels in both nonTg and α6L9’S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by ACh plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9’S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9’S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) compared to nonTg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and norepinephrine (NE) were unchanged in α6L9’S compared to nonTg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9’S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. PMID:24266758

  15. Inactivity–Induced Increase in nAChRs Up–Regulates Shal K+ Channels to Stabilize Synaptic Potentials

    PubMed Central

    Ping, Yong; Tsunoda, Susan

    2011-01-01

    Long–term synaptic changes, which are essential for learning and memory, are dependent on homeostatic mechanisms that stabilize neural activity. Homeostatic responses have also been implicated in pathological conditions, including nicotine addiction. Although multiple homeostatic pathways have been described, little is known about how compensatory responses are tuned to prevent them from overshooting their optimal range of activity. We show that prolonged inhibition of nicotinic acetylcholine receptors (nAChRs), the major excitatory receptor in the Drosophila CNS, results in a homeostatic increase in the Dα7 nAChR. This response then induces an increase in the transient A–type K+ current carried by Shal/Kv4 channels. While increasing Dα7 boosts mEPSCs, the ensuing increase in Shal channels serves to stabilize postsynaptic potentials. This identifies a novel mechanism to fine–tune the homeostatic response. PMID:22081160

  16. Expression patterns of nicotinic subunits α2, α7, α8, and β1 affect the kinetics and pharmacology of ACh-induced currents in adult bee olfactory neuropiles.

    PubMed

    Dupuis, Julien Pierre; Gauthier, Monique; Raymond-Delpech, Valérie

    2011-10-01

    Acetylcholine (ACh) is the main excitatory neurotransmitter of the insect brain, where nicotinic acetylcholine receptors (nAChRs) mediate fast cholinergic synaptic transmission. In the honeybee Apis mellifera, nAChRs are expressed in diverse structures including the primary olfactory centers of the brain, the antennal lobes (ALs) and the mushroom bodies (MBs), where they participate in olfactory information processing. To understand the nature and properties of the nAChRs involved in these processes, we performed a pharmacological and molecular characterization of nAChRs on cultured Kenyon cells of the MBs, using whole cell patch-clamp recordings combined with single-cell RT-PCR. In all cells, applications of ACh as well as nicotinic agonists such as nicotine and imidacloprid induced inward currents with fast desensitization. These currents were fully blocked by saturating doses of the antagonists α-bungarotoxin (α-BGT), dihydroxy-β-erythroidine (DHE), and methyllycaconitine (MLA) (MLA ≥ α-BGT ≥ DHE). Molecular analysis of ACh-responding cells revealed that of the 11 nicotinic receptor subunits encoded within the honeybee genome, α2, α8, and β1 subunits were expressed in adult Kenyon cells. Comparison with the expression pattern of adult AL cells revealed the supplementary presence of subunit α7, which could be responsible for the kinetic and pharmacological differences observed when comparing ACh-induced currents from AL and Kenyon cells. Together, our data demonstrate the existence of functional nAChRs on adult MB Kenyon cells that differ from nAChRs on AL cells in both their molecular composition and pharmacological properties, suggesting that changing receptor subsets could mediate different processing functions depending on the brain structure within the olfactory pathway. PMID:21734106

  17. Nicotinic acetylcholine receptors and cancer

    PubMed Central

    DANG, NINGNING; MENG, XIANGUANG; SONG, HAIYAN

    2016-01-01

    Nicotine, the primary addictive constituent of cigarettes, is believed to contribute to cancer promotion and progression through the activation of nicotinic acetylcholine receptors (nAChRs), which are membrane ligand-gated cation channels. nAChRs activation can be triggered by the neurotransmitter Ach, or certain other biological compounds, such as nicotine. In recent years, genome-wide association studies have indicated that allelic variation in the α5-α3-β4 nAChR cluster on chromosome 15q24-15q25.1 is associated with lung cancer risk. The role of nAChRs in other types of cancer has also been reported. The present review highlights the role of nAChRs in types of human cancer. PMID:27123240

  18. Fluoxetine is neuroprotective in slow-channel congenital myasthenic syndrome.

    PubMed

    Zhu, Haipeng; Grajales-Reyes, Gary E; Alicea-Vázquez, Vivianette; Grajales-Reyes, Jose G; Robinson, KaReisha; Pytel, Peter; Báez-Pagán, Carlos A; Lasalde-Dominicci, Jose A; Gomez, Christopher M

    2015-08-01

    The slow-channel congenital myasthenic syndrome (SCS) is an inherited neurodegenerative disease that caused mutations in the acetylcholine receptor (AChR) affecting neuromuscular transmission. Leaky AChRs lead to Ca(2+) overload and degeneration of the neuromuscular junction (NMJ) attributed to activation of cysteine proteases and apoptotic changes of synaptic nuclei. Here we use transgenic mouse models expressing two different mutations found in SCS to demonstrate that inhibition of prolonged opening of mutant AChRs using fluoxetine not only improves motor performance and neuromuscular transmission but also prevents Ca(2+) overload, the activation of cysteine proteases, calpain, caspase-3 and 9 at endplates, and as a consequence, reduces subsynaptic DNA damage at endplates, suggesting a long term benefit to therapy. These studies suggest that prolonged treatment of SCS patients with open ion channel blockers that preferentially block mutant AChRs is neuroprotective. PMID:25448156

  19. Caenorhabditis elegans nicotinic acetylcholine receptors are required for nociception

    PubMed Central

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

    2014-01-01

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

  20. Development of M1 mAChR Allosteric and Bitopic Ligands: Prospective Therapeutics for the Treatment of Cognitive Deficits

    PubMed Central

    2013-01-01

    Since the cholinergic hypothesis of memory dysfunction was first reported, extensive research efforts have focused on elucidating the mechanisms by which this intricate system contributes to the regulation of processes such as learning, memory, and higher executive function. Several cholinergic therapeutic targets for the treatment of cognitive deficits, psychotic symptoms, and the underlying pathophysiology of neurodegenerative disorders, such as Alzheimer’s disease and schizophrenia, have since emerged. Clinically approved drugs now exist for some of these targets; however, they all may be considered suboptimal therapeutics in that they produce undesirable off-target activity leading to side effects, fail to address the wide variety of symptoms and underlying pathophysiology that characterize these disorders, and/or afford little to no therapeutic effect in subsets of patient populations. A promising target for which there are presently no approved therapies is the M1 muscarinic acetylcholine receptor (M1 mAChR). Despite avid investigation, development of agents that selectively activate this receptor via the orthosteric site has been hampered by the high sequence homology of the binding site between the five muscarinic receptor subtypes and the wide distribution of this receptor family in both the central nervous system (CNS) and the periphery. Hence, a plethora of ligands targeting less structurally conserved allosteric sites of the M1 mAChR have been investigated. This Review aims to explain the rationale behind allosterically targeting the M1 mAChR, comprehensively summarize and critically evaluate the M1 mAChR allosteric ligand literature to date, highlight the challenges inherent in allosteric ligand investigation that are impeding their clinical advancement, and discuss potential methods for resolving these issues. PMID:23659787

  1. Direct measurement of ACh release from exposed frog nerve terminals: constraints on interpretation of non-quantal release.

    PubMed Central

    Grinnell, A D; Gundersen, C B; Meriney, S D; Young, S H

    1989-01-01

    1. Acetylcholine (ACh) release from enzymatically exposed frog motor nerve terminals has been measured directly with closely apposed outside-out clamped patches of Xenopus myocyte membrane, rich in ACh receptor channels. When placed close to the synaptic surface of the terminal, such a membrane patch detects both nerve-evoked patch currents (EPCs) and spontaneous quantal 'miniature' patch currents (MPCs), from a few micrometres length of the terminal, in response to ACh release from the nearest three to five active zones. 2. Chemical measurements of ACh efflux from whole preparations revealed a spontaneous release rate of 4.1 pmol (2 h)-1, and no significant difference in resting efflux between enzyme-treated and control preparations. The ratio of enzyme-treated to contralateral control muscle efflux averaged 1.17, indicating that enzyme treatment did not affect spontaneous ACh release. Vesamicol (1.7 microM), which blocks the ACh transporter in synaptic vesicles, decreased the spontaneous release of ACh to 67% of control. 3. In the absence of nerve stimulation, the frequency of single-channel openings recorded by outside-out patch probes adjacent to nerve terminals was very low (1-2 min-1), and little different at a distance of hundreds of micrometres, suggesting that if ACh was continually leaking from the terminal in a non-quantal fashion, the amount being released near active zone regions on the terminal was below the limit of detection with the patches. 4. Direct measurements of the sensitivity of the patches, coupled with calculated ACh flux rates, lead to the conclusion that the amount of ACh released non-quantally from the synaptic surface of the frog nerve terminal is less than one-tenth the amount expected if all non-quantal release is from this region of the terminal membrane. 5. Following a series of single nerve shocks or a 50 Hz train of nerve stimuli, the frequency of asynchronous single-channel openings increased for several seconds. This transient

  2. Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms.

    PubMed

    Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G

    2016-01-01

    Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein. PMID:27574787

  3. Acetylcholinesterase Regulates Skeletal In Ovo Development of Chicken Limbs by ACh-Dependent and -Independent Mechanisms

    PubMed Central

    Spieker, Janine; Ackermann, Anica; Salfelder, Anika; Vogel-Höpker, Astrid; Layer, Paul G.

    2016-01-01

    Formation of the vertebrate limb presents an excellent model to analyze a non-neuronal cholinergic system (NNCS). Here, we first analyzed the expression of acetylcholinesterase (AChE) by IHC and of choline acetyltransferase (ChAT) by ISH in developing embryonic chicken limbs (stages HH17-37). AChE outlined formation of bones, being strongest at their distal tips, and later also marked areas of cell death. At onset, AChE and ChAT were elevated in two organizing centers of the limb anlage, the apical ectodermal ridge (AER) and zone of polarizing activity (ZPA), respectively. Thereby ChAT was expressed shortly after AChE, thus strongly supporting a leading role of AChE in limb formation. Then, we conducted loss-of-function studies via unilateral implantation of beads into chicken limb anlagen, which were soaked in cholinergic components. After varying periods, the formation of cartilage matrix and of mineralizing bones was followed by Alcian blue (AB) and Alizarin red (AR) stainings, respectively. Both acetylcholine (ACh)- and ChAT-soaked beads accelerated bone formation in ovo. Notably, inhibition of AChE by BW284c51, or by the monoclonal antibody MAB304 delayed cartilage formation. Since bead inhibition of BChE was mostly ineffective, an ACh-independent action during BW284c51 and MAB304 inhibition was indicated, which possibly could be due to an enzymatic side activity of AChE. In conclusion, skeletogenesis in chick is regulated by an ACh-dependent cholinergic system, but to some extent also by an ACh-independent aspect of the AChE protein. PMID:27574787

  4. Sesquiterpenes and a monoterpenoid with acetylcholinesterase (AchE) inhibitory activity from Valeriana officinalis var. latiofolia in vitro and in vivo.

    PubMed

    Chen, Heng-Wen; He, Xuan-Hui; Yuan, Rong; Wei, Ben-Jun; Chen, Zhong; Dong, Jun-Xing; Wang, Jie

    2016-04-01

    Acetylcholinesterase Inhibitor (AchEI) is the most extensive in all anti-dementia drugs. The extracts and isolated compounds from the Valeriana genus have shown anti-dementia bioactivity. Four new sesquiterpenoids (1-4) and a new monoterpenoid (5) were isolated from the root of Valeriana officinalis var. latiofolia. The acetylcholinesterase (AchE) inhibitory activity of isolates was evaluated by modified Ellman method in vitro. Learning and memory ability of compound 4 on mice was evaluated by the Morris water maze. The contents of acetylcholine (Ach), acetylcholine transferase (ChAT) and AchE in mice brains were determined by colorimetry. The results showed IC50 of compound 4 was 0.161 μM in vitro. Compared with the normal group, the learning and memory ability of mice and the contents of Ach and ChAT decreased in model group mice (P<0.01), while the AchE increased (P<0.01). Compared with the model group, Ach and ChAT in the positive control group, the high-dose group and the medium-dose group increased (P<0.01), while the AchE decreased (P<0.01). Compound 4 can improve the learning and memory abilities of APPswe/PSΔE9 double-transgenic mice, and the mechanism may be related to the regulation of the relative enzyme in the cholinergic system. PMID:26976216

  5. GABA interneurons mediate the rapid antidepressant-like effects of scopolamine.

    PubMed

    Wohleb, Eric S; Wu, Min; Gerhard, Danielle M; Taylor, Seth R; Picciotto, Marina R; Alreja, Meenakshi; Duman, Ronald S

    2016-07-01

    Major depressive disorder (MDD) is a recurring psychiatric illness that causes substantial health and socioeconomic burdens. Clinical reports have revealed that scopolamine, a nonselective muscarinic acetylcholine receptor antagonist, produces rapid antidepressant effects in individuals with MDD. Preclinical models suggest that these rapid antidepressant effects can be recapitulated with blockade of M1-type muscarinic acetylcholine receptors (M1-AChR); however, the cellular mechanisms underlying activity-dependent synaptic and behavioral responses to scopolamine have not been determined. Here, we demonstrate that the antidepressant-like effects of scopolamine are mediated by GABA interneurons in the medial prefrontal cortex (mPFC). Both GABAergic (GAD67+) interneurons and glutamatergic (CaMKII+) interneurons in the mPFC expressed M1-AChR. In mice, viral-mediated knockdown of M1-AChR specifically in GABAergic neurons, but not glutamatergic neurons, in the mPFC attenuated the antidepressant-like effects of scopolamine. Immunohistology and electrophysiology showed that somatostatin (SST) interneurons in the mPFC express M1-AChR at higher levels than parvalbumin interneurons. Moreover, knockdown of M1-AChR in SST interneurons in the mPFC demonstrated that M1-AChR expression in these neurons is required for the rapid antidepressant-like effects of scopolamine. These data indicate that SST interneurons in the mPFC are a promising pharmacological target for developing rapid-acting antidepressant therapies. PMID:27270172

  6. Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes.

    PubMed

    Inoue, Tsuyoshi; Abe, Chikara; Sung, Sun-Sang J; Moscalu, Stefan; Jankowski, Jakub; Huang, Liping; Ye, Hong; Rosin, Diane L; Guyenet, Patrice G; Okusa, Mark D

    2016-05-01

    The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes. PMID:27088805

  7. Vagus nerve stimulation mediates protection from kidney ischemia-reperfusion injury through α7nAChR+ splenocytes

    PubMed Central

    Inoue, Tsuyoshi; Abe, Chikara; Sung, Sun-sang J.; Moscalu, Stefan; Jankowski, Jakub; Huang, Liping; Ye, Hong; Guyenet, Patrice G.

    2016-01-01

    The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes. PMID:27088805

  8. New insights on the molecular recognition of imidacloprid with Aplysia californica AChBP: a computational study.

    PubMed

    Cerón-Carrasco, José P; Jacquemin, Denis; Graton, Jérôme; Thany, Steeve; Le Questel, Jean-Yves

    2013-04-18

    The binding of imidacloprid (IMI), the forerunner of neonicotinoid insecticides, with the acetylcholine binding protein (AChBP) from Aplysia californica, the established model for the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a two-layer ONIOM partition approach (M06-2X/6-311G(d):PM6). Our calculations allow delineating the contributions of the key residues of AChBP for IMI binding. In particular, the importance of Trp147 and Cys190-191, through weak CH···π interactions and both van der Waals and hydrogen-bond (H-bond) interactions, respectively, are highlighted. Furthermore, H-bonds between hydroxyl groups of both Ser189 and Tyr55 and the IMI nitro group are pointed out. The participation of Ile118, whose main chain NH and carbonyl group are hydrogen-bonded with the IMI pyridinic nitrogen through a water molecule, is characterized. Our simulations also indicate the presence of a significant contribution of this residue through van der Waals interactions. The various trends obtained by the calculations of the pairwise interaction energies are confirmed through a complementary noncovalent interaction (NCI) analysis of selected IMI-AChBP amino acid pairs. Indeed, the contribution of a halogen-bond interaction between IMI and AChBP, recently proposed in the literature, is corroborated by our NCI analysis. PMID:23521537

  9. Highly selective and sensitive detection of neurotransmitters using receptor-modified single-walled carbon nanotube sensors

    NASA Astrophysics Data System (ADS)

    Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2013-07-01

    We present receptor-modified carbon nanotube sensors for the highly selective and sensitive detection of acetylcholine (ACh), one kind of neurotransmitter. Here, we successfully expressed the M1 muscarinic acetylcholine receptor (M1 mAChR), a family of G protein-coupled receptors (GPCRs), in E. coli and coated single-walled carbon nanotube (swCNT)-field effect transistors (FETs) with lipid membrane including the receptor, enabling highly selective and sensitive ACh detection. Using this sensor, we could detect ACh at 100 pM concentration. Moreover, we showed that this sensor could selectively detect ACh among other neurotransmitters. This is the first demonstration of the real-time detection of ACh using specific binding between ACh and M1 mAChR, and it may lead to breakthroughs for various applications such as disease diagnosis and drug screening.

  10. Identification and pharmacological characterization of 3,6-diazabicyclo[3.1.1]heptane-3-carboxamides as novel ligands for the α4β2 and α6/α3β2β3 nicotinic acetylcholine receptors (nAChRs).

    PubMed

    Strachan, Jon-Paul; Kombo, David C; Mazurov, Anatoly; Heemstra, Ronald; Bhatti, Balwinder S; Akireddy, Rao; Murthy, Srinivasa; Miao, Lan; Jett, John E; Speake, Jason; Bencherif, Merouane

    2014-10-30

    We have synthesized a novel series of compounds, 3,6-diazabicyclo[3.1.1]heptane-3-carboxamides, targeting both the α4β2 and α6/α3β2β3 nAChRs. Members of the obtained chemical library are partial or full agonists at both the high sensitivity (α4)2(β2)3 and α6/α3β2β3 nAChRs. 3-(Cyclopropylcarbonyl)-3,6-diazabicyclo[3.1.1]heptane (TC-8831 or compound 7 herein) demonstrated a safe in vitro pharmacological profile and the potential for reducing or preventing L-dopa-induced dyskinesias (LID) in several in vivo animal models [1-4]. In vivo metabolism studies in rat and in vitro metabolism studies in liver microsomes from human, rat, dog and monkey showed TC-8831 to be relatively stable. In vivo pharmacokinetic analysis in the rat confirmed brain penetration, with an average brain:plasma ratio of approximately 0.3 across time points from 0.5 to 4 h. Docking into homology models predicted alternative binding modes for TC-8831 and highlighted the importance of the cationic center, hydrogen-bond acceptor, and hydrophobic aliphatic features in promoting binding affinity to both nAChRs. Pharmacophore elucidation confirmed the importance of these key interactions. QSAR modeling suggested that binding affinity is primarily driven by ligand shape, relative positive charge distribution onto the molecular surface, and molecular flexibility. Of the two subtypes, ligand binding to α6β2β3 appears to be more sensitive to bulkiness and flexibility. PMID:25147147

  11. Cardanol-derived AChE inhibitors: Towards the development of dual binding derivatives for Alzheimer's disease.

    PubMed

    Lemes, Laís Flávia Nunes; de Andrade Ramos, Giselle; de Oliveira, Andressa Souza; da Silva, Fernanda Motta R; de Castro Couto, Gina; da Silva Boni, Marina; Guimarães, Marcos Jorge R; Souza, Isis Nem O; Bartolini, Manuela; Andrisano, Vincenza; do Nascimento Nogueira, Patrícia Coelho; Silveira, Edilberto Rocha; Brand, Guilherme D; Soukup, Ondřej; Korábečný, Jan; Romeiro, Nelilma C; Castro, Newton G; Bolognesi, Maria Laura; Romeiro, Luiz Antonio Soares

    2016-01-27

    Cardanol is a phenolic lipid component of cashew nut shell liquid (CNSL), obtained as the byproduct of cashew nut food processing. Being a waste product, it has attracted much attention as a precursor for the production of high-value chemicals, including drugs. On the basis of these findings and in connection with our previous studies on cardanol derivatives as acetylcholinesterase (AChE) inhibitors, we designed a novel series of analogues by including a protonable amino moiety belonging to different systems. Properly addressed docking studies suggested that the proposed structural modifications would allow the new molecules to interact with both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE, thus being able to act as dual binding inhibitors. To disclose whether the new molecules showed the desired profile, they were first tested for their cholinesterase inhibitory activity towards EeAChE and eqBuChE. Compound 26, bearing an N-ethyl-N-(2-methoxybenzyl)amine moiety, showed the highest inhibitory activity against EeAChE, with a promising IC50 of 6.6 μM, and a similar inhibition profile of the human isoform (IC50 = 5.7 μM). As another positive feature, most of the derivatives did not show appreciable toxicity against HT-29 cells, up to a concentration of 100 μM, which indicates drug-conform behavior. Also, compound 26 is capable of crossing the blood-brain barrier (BBB), as predicted by a PAMPA-BBB assay. Collectively, the data suggest that the approach to obtain potential anti-Alzheimer drugs from CNSL is worth of further pursuit and development. PMID:26735910

  12. Design, Synthesis, and Evaluation of Donepezil-Like Compounds as AChE and BACE-1 Inhibitors.

    PubMed

    Costanzo, Paola; Cariati, Luca; Desiderio, Doriana; Sgammato, Roberta; Lamberti, Anna; Arcone, Rosaria; Salerno, Raffaele; Nardi, Monica; Masullo, Mariorosario; Oliverio, Manuela

    2016-05-12

    An ecofriendly synthetic pathway for the synthesis of donepezil precursors is described. Alternative energy sources were used for the total synthesis in order to improve yields, regioselectively, and rate of each synthetic step and to reduce the coproduction of waste at the same time. For all products, characterized by an improved structural rigidity respect to donepezil, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1, and the effect on SHSY-5Y neuroblastoma cells viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer's disease were envisaged. PMID:27190595

  13. Nicotine Inhibits Cisplatin-Induced Apoptosis via Regulating α5-nAChR/AKT Signaling in Human Gastric Cancer Cells.

    PubMed

    Jia, Yanfei; Sun, Haiji; Wu, Hongqiao; Zhang, Huilin; Zhang, Xiuping; Xiao, Dongjie; Ma, Xiaoli; Wang, Yunshan

    2016-01-01

    Gastric cancer incidence demonstrates a strong etiologic association with smoking. Nicotine, the major component in tobacco, is a survival agonist that inhibits apoptosis induced by certain chemotherapeutic agents, but the precise mechanisms involved remain largely unknown. Recently studies have indicated that α5-nicotinic acetylcholine receptor (α5-nAChR) is highly associated with lung cancer risk and nicotine dependence. Nevertheless, no information has been available about whether nicotine also affects proliferation of human gastric cancer cells through regulation of α5-nAChR. To evaluate the hypothesis that α5-nAChR may play a role in gastric cancer, we investigated its expression in gastric cancer tissues and cell lines. The expression of α5-nAChR increased in gastric cancer tissue compared with para-carcinoma tissues. In view of the results, we proceeded to investigate whether nicotine inhibits cisplatin-induced apoptosis via regulating α5-nAChR in gastric cancer cell. The results showed that nicotine significantly promoted cell proliferation in a dose and time-dependent manner through α5-nAChR activation in human gastric cells. Furthermore, nicotine inhibited apoptosis induced by cisplatin. Silence of α5-nAChR ablated the protective effects of nicotine. However, when co-administrating LY294002, an inhibitor of PI3K/AKT pathway, an increased apoptosis was observed. This effect correlated with the induction of Bcl-2, Bax, Survivin and Caspase-3 by nicotine in gastric cell lines. These results suggest that exposure to nicotine might negatively impact the apoptotic potential of chemotherapeutic drugs and that α5-nAChR/AKT signaling plays a key role in the anti-apoptotic activity of nicotine induced by cisplatin. PMID:26909550

  14. The Ache: Genocide Continues in Paraguay. IWGIA Document No. 17.

    ERIC Educational Resources Information Center

    Munzel, Mark

    In 1972, the Paraguayan Roman Catholic Church protested against the massacre of Indians in Paraguay. This was followed by further protests from Paraguayan intellectuals. These protests led to the removal of Jesus de Pereira, one of the executors of the official Ache policy. Thus, the critics were appeased. Since the beginning of 1973, new protests…

  15. Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

    PubMed Central

    Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2013-01-01

    We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

  16. Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.

    PubMed

    Padilla-Morales, Luis F; Colón-Sáez, José O; González-Nieves, Joel E; Quesada-González, Orestes; Lasalde-Dominicci, José A

    2016-01-01

    In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyte plasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex

  17. Diagnosis of myasthenia gravis: Comparison of anti-nicotinic acetyl choline receptor antibodies, repetitive nerve stimulation and Neostigmine tests at a tertiary neuro care centre in India, a ten year study.

    PubMed

    Patil, Shripad A; Bokoliya, Suresh C; Nagappa, Madhu; Taly, Arun B

    2016-03-15

    Anti-nicotinic AChR antibodies (Anti-nAChR antibodies), Repetitive Nerve Stimulation (RNS) and Neostigmine test are used for diagnosis of myasthenia gravis (MG). We compared their diagnostic agreement in a cohort of 486 MG patients over a period of ten years. Anti-nAChR antibodies, RNS and Neostigmine test showed positivity of 57.36%, 51.78%, and 93.4% respectively in ocular myasthenia and 93.77%, 82.35%, and 97.92% respectively in generalized myasthenia group. Neostigmine test showed higher positivity than anti-nAChR antibodies and RNS test in both groups. A marginal to fair agreement was observed between these tests highlighting their significance in the diagnosis of the disease. PMID:26943963

  18. Insect nicotinic receptor interactions in vivo with neonicotinoid, organophosphorus, and methylcarbamate insecticides and a synergist

    PubMed Central

    Shao, Xusheng; Xia, Shanshan; Durkin, Kathleen A.; Casida, John E.

    2013-01-01

    The nicotinic acetylcholine (ACh) receptor (nAChR) is the principal insecticide target. Nearly half of the insecticides by number and world market value are neonicotinoids acting as nAChR agonists or organophosphorus (OP) and methylcarbamate (MC) acetylcholinesterase (AChE) inhibitors. There was no previous evidence for in vivo interactions of the nAChR agonists and AChE inhibitors. The nitromethyleneimidazole (NMI) analog of imidacloprid, a highly potent neonicotinoid, was used here as a radioligand, uniquely allowing for direct measurements of house fly (Musca domestica) head nAChR in vivo interactions with various nicotinic agents. Nine neonicotinoids inhibited house fly brain nAChR [3H]NMI binding in vivo, corresponding to their in vitro potency and the poisoning signs or toxicity they produced in intrathoracically treated house flies. Interestingly, nine topically applied OP or MC insecticides or analogs also gave similar results relative to in vivo nAChR binding inhibition and toxicity, but now also correlating with in vivo brain AChE inhibition, indicating that ACh is the ultimate OP- or MC-induced nAChR active agent. These findings on [3H]NMI binding in house fly brain membranes validate the nAChR in vivo target for the neonicotinoids, OPs and MCs. As an exception, the remarkably potent OP neonicotinoid synergist, O-propyl O-(2-propynyl) phenylphosphonate, inhibited nAChR in vivo without the corresponding AChE inhibition, possibly via a reactive ketene metabolite reacting with a critical nucleophile in the cytochrome P450 active site and the nAChR NMI binding site. PMID:24108354

  19. The role of antibodies in myasthenia gravis.

    PubMed

    De Baets, M; Stassen, M H W

    2002-10-15

    Myasthenia gravis is an autoimmune disease associated with antibodies directed to the postsynaptic acetylcholine receptor. These antibodies reduce the number of receptors. Autoantibodies against AChR and other muscle antigens can be used for the diagnosis of myasthenia gravis and related disorders. The origin and the role of these antibodies in the disease are discussed. Experimental autoimmune myasthenia gravis, an experimental model closely mimicking the disease, has provided answers to many questions about the role of antibodies, complement macrophages and AChR anchor proteins. Genetically modified anti-AChR antibodies may also be used in the future to treat myasthenia. PMID:12220686

  20. Enhanced synthesis and release of dopamine in transgenic mice with gain-of-function α6* nAChRs.

    PubMed

    Wang, Yuexiang; Lee, Jang-Won; Oh, Gyeon; Grady, Sharon R; McIntosh, J Michael; Brunzell, Darlene H; Cannon, Jason R; Drenan, Ryan M

    2014-04-01

    α6β2* nicotinic acetylcholine receptors (nAChRs)s in the ventral tegmental area to nucleus accumbens (NAc) pathway are implicated in the response to nicotine, and recent work suggests these receptors play a role in the rewarding action of ethanol. Here, we studied mice expressing gain-of-function α6β2* nAChRs (α6L9'S mice) that are hypersensitive to nicotine and endogenous acetylcholine. Evoked extracellular dopamine (DA) levels were enhanced in α6L9'S NAc slices compared to control, non-transgenic (non-Tg) slices. Extracellular DA levels in both non-Tg and α6L9'S slices were further enhanced in the presence of GBR12909, suggesting intact DA transporter function in both mouse strains. Ongoing α6β2* nAChR activation by acetylcholine plays a role in enhancing DA levels, as α-conotoxin MII completely abolished evoked DA release in α6L9'S slices and decreased spontaneous DA release from striatal synaptosomes. In HPLC experiments, α6L9'S NAc tissue contained significantly more DA, 3,4-dihydroxyphenylacetic acid, and homovanillic acid compared to non-Tg NAc tissue. Serotonin (5-HT), 5-hydroxyindoleacetic acid, and norepinephrine (NE) were unchanged in α6L9'S compared to non-Tg tissue. Western blot analysis revealed increased tyrosine hydroxylase expression in α6L9'S NAc. Overall, these results show that enhanced α6β2* nAChR activity in NAc can stimulate DA production and lead to increased extracellular DA levels. PMID:24266758

  1. Nicotinic Receptors in Neurodegeneration

    PubMed Central

    Posadas, Inmaculada; López-Hernández, Beatriz; Ceña, Valentín

    2013-01-01

    Many studies have focused on expanding our knowledge of the structure and diversity of peripheral and central nicotinic receptors. Nicotinic acetylcholine receptors (nAChRs) are members of the Cys-loop superfamily of pentameric ligand-gated ion channels, which include GABA (A and C), serotonin, and glycine receptors. Currently, 9 alpha (α2-α10) and 3 beta (β2-β4) subunits have been identified in the central nervous system (CNS), and these subunits assemble to form a variety of functional nAChRs. The pentameric combination of several alpha and beta subunits leads to a great number of nicotinic receptors that vary in their properties, including their sensitivity to nicotine, permeability to calcium and propensity to desensitize. In the CNS, nAChRs play crucial roles in modulating presynaptic, postsynaptic, and extrasynaptic signaling, and have been found to be involved in a complex range of CNS disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), schizophrenia, Tourette´s syndrome, anxiety, depression and epilepsy. Therefore, there is growing interest in the development of drugs that modulate nAChR functions with optimal benefits and minimal adverse effects. The present review describes the main characteristics of nAChRs in the CNS and focuses on the various compounds that have been tested and are currently in phase I and phase II trials for the treatment of neurodegenerative diseases including PD, AD and age-associated memory and mild cognitive impairment. PMID:24179465

  2. PACAP induces plasticity at autonomic synapses by nAChR-dependent NOS1 activation and AKAP-mediated PKA targeting.

    PubMed

    Jayakar, Selwyn S; Pugh, Phyllis C; Dale, Zack; Starr, Eric R; Cole, Samantha; Margiotta, Joseph F

    2014-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide found at synapses throughout the central and autonomic nervous system. We previously found that PACAP engages a selective G-protein coupled receptor (PAC1R) on ciliary ganglion neurons to rapidly enhance quantal acetylcholine (ACh) release from presynaptic terminals via neuronal nitric oxide synthase (NOS1) and cyclic AMP/protein kinase A (PKA) dependent processes. Here, we examined how PACAP stimulates NO production and targets resultant outcomes to synapses. Scavenging extracellular NO blocked PACAP-induced plasticity supporting a retrograde (post- to presynaptic) NO action on ACh release. Live-cell imaging revealed that PACAP stimulates NO production by mechanisms requiring NOS1, PKA and Ca(2+) influx. Ca(2+)-permeable nicotinic ACh receptors composed of α7 subunits (α7-nAChRs) are potentiated by PKA-dependent PACAP/PAC1R signaling and were required for PACAP-induced NO production and synaptic plasticity since both outcomes were drastically reduced following their selective inhibition. Co-precipitation experiments showed that NOS1 associates with α7-nAChRs, many of which are perisynaptic, as well as with heteromeric α3*-nAChRs that generate the bulk of synaptic activity. NOS1-nAChR physical association could facilitate NO production at perisynaptic and adjacent postsynaptic sites to enhance focal ACh release from juxtaposed presynaptic terminals. The synaptic outcomes of PACAP/PAC1R signaling are localized by PKA anchoring proteins (AKAPs). PKA regulatory-subunit overlay assays identified five AKAPs in ganglion lysates, including a prominent neuronal subtype. Moreover, PACAP-induced synaptic plasticity was selectively blocked when PKA regulatory-subunit binding to AKAPs was inhibited. Taken together, our findings indicate that PACAP/PAC1R signaling coordinates nAChR, NOS1 and AKAP activities to induce targeted, retrograde plasticity at autonomic synapses. Such

  3. Assessment of the expression and role of the α1-nAChR subunit in efferent cholinergic function during the development of the mammalian cochlea.

    PubMed

    Roux, Isabelle; Wu, Jingjing Sherry; McIntosh, J Michael; Glowatzki, Elisabeth

    2016-08-01

    Hair cell (HC) activity in the mammalian cochlea is modulated by cholinergic efferent inputs from the brainstem. These inhibitory inputs are mediated by calcium-permeable nicotinic acetylcholine receptors (nAChRs) containing α9- and α10-subunits and by subsequent activation of calcium-dependent potassium channels. Intriguingly, mRNAs of α1- and γ-nAChRs, subunits of the "muscle-type" nAChR have also been found in developing HCs (Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. J Neurosci 35: 5870-5883, 2015; Scheffer D, Sage C, Plazas PV, Huang M, Wedemeyer C, Zhang DS, Chen ZY, Elgoyhen AB, Corey DP, Pingault V. J Neurochem 103: 2651-2664, 2007; Sinkkonen ST, Chai R, Jan TA, Hartman BH, Laske RD, Gahlen F, Sinkkonen W, Cheng AG, Oshima K, Heller S. Sci Rep 1: 26, 2011) prompting proposals that another type of nAChR is present and may be critical during early synaptic development. Mouse genetics, histochemistry, pharmacology, and whole cell recording approaches were combined to test the role of α1-nAChR subunit in HC efferent synapse formation and cholinergic function. The onset of α1-mRNA expression in mouse HCs was found to coincide with the onset of the ACh response and efferent synaptic function. However, in mouse inner hair cells (IHCs) no response to the muscle-type nAChR agonists (±)-anatoxin A, (±)-epibatidine, (-)-nicotine, or 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) was detected, arguing against the presence of an independent functional α1-containing muscle-type nAChR in IHCs. In α1-deficient mice, no obvious change of IHC efferent innervation was detected at embryonic day 18, contrary to the hyperinnervation observed at the neuromuscular junction. Additionally, ACh response and efferent synaptic activity were detectable in α1-deficient IHCs, suggesting that α1 is not necessary for assembly and membrane targeting of nAChRs or for efferent synapse formation in IHCs. PMID:27098031

  4. The role of α7 nicotinic acetylcholine receptor in modulation of heart rate dynamics in endotoxemic rats.

    PubMed

    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. Acetylcholinesterase (AChE) and heat shock proteins (Hsp70) of gypsy moth (Lymantria dispar L.) larvae in response to long-term fluoranthene exposure.

    PubMed

    Mrdaković, Marija; Ilijin, Larisa; Vlahović, Milena; Matić, Dragana; Gavrilović, Anja; Mrkonja, Aleksandra; Perić-Mataruga, Vesna

    2016-09-01

    Polycyclic aromatic hydrocarbons (PAHs) may affect biochemical and physiological processes in living organisms, thus impairing fitness related traits and influencing their populations. This imposes the need for providing early-warning signals of pollution. Our study aimed to examine changes in the activity of acetylcholinesterase (AChE) and the concentration of heat shock proteins (Hsp70) in homogenates of brain tissues of fifth instar gypsy moth (Lymantria dispar L.) larvae, exposed to the ubiquitous PAH, fluoranthene, supplemented to the rearing diet. Significantly increased activity of AChE in larvae fed on the diets with high fluoranthene concentrations suggests the necessity for elucidation of the role of AChE in these insects when exposed to PAH pollution. Significant induction of Hsp70 in gypsy moth larvae reared on the diets containing low fluoranthene concentrations, indicate that changes in the level of Hsp70 might be useful as an indicator of pollution in this widespread forest species. PMID:27343862

  6. 77 FR 40148 - Proposed Collection of Information: ACH Vendor/Miscellaneous Payment Enrollment Form

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-06

    ... Fiscal Service Proposed Collection of Information: ACH Vendor/Miscellaneous Payment Enrollment Form... comments concerning the SF 3881 ``ACH Vendor/Miscellaneous Payment Enrollment Form.'' DATES: Written... solicits comments on the collection of information described below: Title: ACH Vendor/Miscellaneous...

  7. Target site insensitivity mutations in the AChE enzyme confer resistance to organophosphorous insecticides in Leptinotarsa decemlineata (Say).

    PubMed

    Malekmohammadi, M; Galehdari, H

    2016-01-01

    In the present study, we demonstrated the use and optimization of the tetra-primer ARMS-PCR procedure to detect and analyze the frequency of the R30K and I392T mutations in resistant field populations of CPB. The R30K mutation was detected in 72%, 84%, 52% and 64% of Bahar, Dehpiaz, Aliabad and Yengijeh populations, respectively. Overall frequencies of the I392T mutation were 12%, 8% and 16% of Bahar, Aliabad and Yengijeh populations, respectively. No I392T point mutation was found among samples from Dehpiaz field population. Moreover, only 31% and 2% of samples from the resistant field populations were homozygous for R30K and I392T mutations, respectively. No individual simultaneously had both I392T and S291G/R30K point mutations. The incidence of individuals with both S291G and R30K point mutations in the samples from Bahar, Dehpiaz, Aliabad, and Yengijeh populations were 31.5%, 44.7%, 41.6%, and 27.3% respectively. Genotypes determined by the tetra-primer ARMS-PCR method were consistent with those determined by PCR sequencing. There was no significant correlation between the mutation frequencies and resistance levels in the resistant populations, indicating that other mutations may contribute to this variation. Polymorphism in the partial L. decemlineata cDNA AChE gene Ldace2 of four field populations was identified by direct sequencing of PCR-amplified fragments. Among 45 novel mutations detected in this study, T29P mutation was found across all four field populations that likely contribute to the AChE insensitivity. Site-directed mutagenesis and protein expression experiments are needed for a more complete evaluation. PMID:26778439

  8. Fluorescence Quenching Determination of Uranium (VI) Binding Properties by Two Functional Proteins: Acetylcholinesterase (AChE) and Vitellogenin (Vtg).

    PubMed

    Coppin, Frédéric; Michon, Jérôme; Garnier, Cédric; Frelon, Sandrine

    2015-05-01

    The interactions between uranium and two functional proteins (AChE and Vtg) were investigated using fluorescence quenching measurements. The combined use of a microplate spectrofluorometer and logarithmic additions of uranium into protein solutions allowed us to define the fluorescence quenching over a wide range of [U]/[Pi] ratios (from 1 to 3235) at physiologically relevant conditions of pH. Results showed that fluorescence from the two functional proteins was quenched by UO2 (2+). Stoichiometry reactions, fluorescence quenching mechanisms and complexing properties of proteins, i.e. binding constants and binding sites densities, were determined using classic fluorescence quenching methods and curve-fitting software (PROSECE). It was demonstrated that in our test conditions, the protein complexation by uranium could be simulated by two specific sites (L1 and L2). The obtained complexation constant values are log K1 = 5.7 (±1.0), log K2 = 4.9 (±1.1); L1 = 83 (±2), L2 = 2220 (±150) for U(VI) - Vtg and log K1 = 8.1 (±0.9), log K2 = 6.6 (±0.5), L1 = 115 (±16), L2 = 530 (±23) for U(VI)-AChE (Li is expressed in mol/mol of protein). PMID:25764300

  9. WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

    PubMed Central

    Yu, Pin; Liu, Shui-Ping; Bu, Qing-Ting; Zhou, Zhen-Xing; Zhu, Zhen-Hong; Huang, Fang-Liang

    2014-01-01

    Detailed mechanisms of WhiB-like (Wbl) proteins involved in antibiotic biosynthesis and morphological differentiation are poorly understood. Here, we characterize the role of WblAch, a Streptomyces chattanoogensis L10 protein belonging to this superfamily. Based on DNA microarray data and verified by real-time quantitative PCR (qRT-PCR), the expression of wblAch was shown to be positively regulated by AdpAch. Gel retardation assays and DNase I footprinting experiments showed that AdpAch has specific DNA-binding activity for the promoter region of wblAch. Gene disruption and genetic complementation revealed that WblAch acts in a positive manner to regulate natamycin production. When wblAch was overexpressed in the wild-type strain, the natamycin yield was increased by ∼30%. This provides a strategy to generate improved strains for natamycin production. Moreover, transcriptional analysis showed that the expression levels of whi genes (including whiA, whiB, whiH, and whiI) were severely depressed in the ΔwblAch mutant, suggesting that WblAch plays a part in morphological differentiation by influencing the expression of the whi genes. PMID:25172865

  10. [Genetic defects and disorders at the neuromuscular junction].

    PubMed

    Ohno, Kinji

    2011-07-01

    Genetic defects in molecules expressed at the neuromuscular junction (NMJ) cause congenital myasthenic syndromes (CMSs), which are characterized by muscle weakness, abnormal fatigability, amyotrophy, and minor facial anomalies. Muscle weakness mostly develops under 2 years but is also sometimes seen in adults. Mutations identified to date include (i) muscle nicotinic acetylcholine receptor (AChR) subunits, (ii) rapsyn that anchors and clusters AChRs at the neuromuscular junction, (iii) agrin that is released from the nerve terminal and induces AChR clustering by stimulating the downstream LRP4/MuSK/Dok-7/rapsyn/AChR pathway, (iv) muscle-specific kinase (MuSK) that transmits the AChR-clustering signal from agrin/LRP4 to rapsyn/AChR, (v) Dok-7 that transmits the AChR-clustering signal from agrin/LRP4/MuSK to rapsyn/AChR, (vi) skeletal muscle sodium channel type 1.4 (Nav1.4) that spreads the depolarization potential from the endplate throughout muscle fibers, (vii) collagen Q that anchors acetylcholinesterase to the synaptic basal lamina, and (viii) choline acetyltransferase that resynthesizes acetylcholine from recycled choline at the nerve terminal. In addition, mutations in the heparin sulfate proteoglycan perlecan, which binds to many molecules including collagen Q and dystroglycan, causes Schwartz-Jampel syndrome. Interestingly, mutations in LRP4 cause Cenani-Lenz syndactyly syndrome but not CMS. AChR, MuSK, and LRP4 are also targets of auto-antibodies in myasthenia gravis. In addition, molecules at the NMJ are targets of many other disease states AChRs are blocked by the snake toxin alpha-bungarotoxin and the plant poison curare. The presynaptic SNARE complex is attacked by botulinum toxin. Acetylcholinesterase is inhibited by the nerve gas sarin and by organophosphate pesticides. This review focuses on the molecular bases underlying defects of AChR, rapsyn, Nav1.4, collagen Q, and choline acetyltransferase. PMID:21747136

  11. Dysfunctional Presynaptic M2 Receptors in the Presence of Chronically High Acetylcholine Levels: Data from the PRiMA Knockout Mouse

    PubMed Central

    Mohr, Franziska; Krejci, Eric; Zimmermann, Martina; Klein, Jochen

    2015-01-01

    The muscarinic M2 receptor (M2R) acts as a negative feedback regulator in central cholinergic systems. Activation of the M2 receptor limits acetylcholine (ACh) release, especially when ACh levels are increased because acetylcholinesterase (AChE) activity is acutely inhibited. Chronically high ACh levels in the extracellular space, however, were reported to down-regulate M2R to various degrees. In the present study, we used the PRiMA knockout mouse which develops severely reduced AChE activity postnatally to investigate ACh release, and we used microdialysis to investigate whether the function of M2R to reduce ACh release in vivo was impaired in adult PRiMA knockout mice. We first show that striatal and hippocampal ACh levels, while strongly increased, still respond to AChE inhibitors. Infusion or injection of oxotremorine, a muscarinic M2 agonist, reduced ACh levels in wild-type mice but did not significantly affect ACh levels in PRiMA knockout mice or in wild-type mice in which ACh levels were artificially increased by infusion of neostigmine. Scopolamine, a muscarinic antagonist, increased ACh levels in wild-type mice receiving neostigmine, but not in wild-type mice or in PRiMA knockout mice. These results demonstrate that M2R are dysfunctional and do not affect ACh levels in PRiMA knockout mice, likely because of down-regulation and/or loss of receptor-effector coupling. Remarkably, this loss of function does not affect cognitive functions in PRiMA knockout mice. Our results are discussed in the context of AChE inhibitor therapy as used in dementia. PMID:26506622

  12. Novel bis-(−)-nor-meptazinol derivatives act as dual binding site AChE inhibitors with metal-complexing property

    SciTech Connect

    Zheng, Wei; Li, Juan; Qiu, Zhuibai; Xia, Zheng; Li, Wei; Yu, Lining; Chen, Hailin; Chen, Jianxing; Chen, Yan; Hu, Zhuqin; Zhou, Wei; Shao, Biyun; Cui, Yongyao; Xie, Qiong; Chen, Hongzhuan

    2012-10-01

    The strategy of dual binding site acetylcholinesterase (AChE) inhibition along with metal chelation may represent a promising direction for multi-targeted interventions in the pathophysiological processes of Alzheimer's disease (AD). In the present study, two derivatives (ZLA and ZLB) of a potent dual binding site AChE inhibitor bis-(−)-nor-meptazinol (bis-MEP) were designed and synthesized by introducing metal chelating pharmacophores into the middle chain of bis-MEP. They could inhibit human AChE activity with IC{sub 50} values of 9.63 μM (for ZLA) and 8.64 μM (for ZLB), and prevent AChE-induced amyloid-β (Aβ) aggregation with IC{sub 50} values of 49.1 μM (for ZLA) and 55.3 μM (for ZLB). In parallel, molecular docking analysis showed that they are capable of interacting with both the catalytic and peripheral anionic sites of AChE. Furthermore, they exhibited abilities to complex metal ions such as Cu(II) and Zn(II), and inhibit Aβ aggregation triggered by these metals. Collectively, these results suggest that ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency, and may be potential leads of value for further study on disease-modifying treatment of AD. -- Highlights: ► Two novel bis-(−)-nor-meptazinol derivatives are designed and synthesized. ► ZLA and ZLB may act as dual binding site AChEIs with metal-chelating potency. ► They are potential leads for disease-modifying treatment of Alzheimer's disease.

  13. Thiochrome enhances acetylcholine affinity at muscarinic M4 receptors: receptor subtype selectivity via cooperativity rather than affinity.

    PubMed

    Lazareno, S; Dolezal, V; Popham, A; Birdsall, N J M

    2004-01-01

    Thiochrome (2,7-dimethyl-5H-thiachromine-8-ethanol), an oxidation product and metabolite of thiamine, has little effect on the equilibrium binding of l-[3H]N-methyl scopolamine ([3H]NMS) to the five human muscarinic receptor subtypes (M1-M5) at concentrations up to 0.3 mM. In contrast, it inhibits [3H]NMS dissociation from M1 to M4 receptors at submillimolar concentrations and from M5 receptors at 1 mM. These results suggest that thiochrome binds allosterically to muscarinic receptors and has approximately neutral cooperativity with [3H]NMS at M1 to M4 and possibly M5 receptors. Thiochrome increases the affinity of acetylcholine (ACh) 3- to 5-fold for inhibiting [3H]NMS binding to M4 receptors but has no effect on ACh affinity at M1 to M3 or M5 receptors. Thiochrome (0.1 mM) also increases the direct binding of [3H]ACh to M4 receptors but decreases it slightly at M2 receptors. In agreement with the binding data, thiochrome does not affect the potency of ACh for stimulating the binding of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) to membranes containing M1 to M3 receptors, but it increases ACh potency 3.5-fold at M4 receptors. It also selectively reduces the release of [3H]ACh from potassium-stimulated slices of rat striatum, which contain autoinhibitory presynaptic M4 receptors, but not from hippocampal slices, which contain presynaptic M2 receptors. We conclude that thiochrome is a selective M4 muscarinic receptor enhancer of ACh affinity and has neutral cooperativity with ACh at M1 to M3 receptors; it therefore demonstrates a powerful new form of selectivity, "absolute subtype selectivity", which is derived from cooperativity rather than from affinity. PMID:14722259

  14. Novel assay utilizing fluorochrome-tagged physostigmine (Ph-F) to in situ detect active acetylcholinesterase (AChE) induced during apoptosis.

    PubMed

    Huang, Xuan; Lee, Brian; Johnson, Gary; Naleway, John; Guzikowski, Anthony; Dai, Wei; Darzynkiewicz, Zbigniew

    2005-01-01

    It was recently reported that acetylcholinesterase (AChE) is expressed in cells undergoing apoptosis and that its presence is essential for assembly of the apoptosome and subsequent caspase-9 activation. To obtain a marker of active AChE that could assay this enzyme in live intact cells and be applicable to fluorescence microscopy and cytometry, the fluorescein-tagged physostigmine (Ph-F), high affinity ligand (inhibitor) reactive with the active center of AChE, was constructed and tested for its ability to in situ label AChE and measure its induction during apoptosis. Ph-F inhibited cholinesterase activity in vitro (IC50 = 10(-6) and 5 x 10(-6) M for equine butyrylcholinesterase and human erythrocyte AChE, respectively) and was a selective marker of cells and structures that were AChE-positive. Thus, exposure of mouse bone marrow cells to Ph-F resulted in the exclusive labeling of megakaryocytes, and of the diaphragm muscle, preferential labeling of the nerve-muscle junctions (end-plates). During apoptosis of carcinoma HeLa cells and leukemic HL-60 or Jurkat cells triggered either by the DNA topoisomerase 1 inhibitor topotecan (TPT) or by oxidative stress (H2O2), the cells become reactive with Ph-F. Their Ph-F derived fluorescence was measured by flow and laser scanning cytometry. The appearance of Ph-F binding sites during apoptosis was preceded by the loss of mitochondrial potential, was concurrent with the presence of activated caspases, and was followed by loss of membrane integrity. At a very early stage of apoptosis, when nucleolar segregation was apparent, the Ph-F binding sites were distinctly localized within the nucleolus and at later stages of apoptosis in the cytoplasm. During apoptosis triggered by TPT, Ph-F binding was preferentially induced in S-phase cells. Our data on megakaryocytes and end-plates indicate that Ph-F reacts with active sites of AChE, and can be used to reveal the presence of this enzyme in live cells and possibly to study its

  15. The use of α-conotoxin ImI to actualize the targeted delivery of paclitaxel micelles to α7 nAChR-overexpressing breast cancer.

    PubMed

    Mei, Dong; Lin, Zhiqiang; Fu, Jijun; He, Bing; Gao, Wei; Ma, Ling; Dai, Wenbing; Zhang, Hua; Wang, Xueqing; Wang, Jiancheng; Zhang, Xuan; Lu, Wanliang; Zhou, Demin; Zhang, Qiang

    2015-02-01

    Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a ligand-gated ion channel, is increasingly emerging as a new tumor target owing to its expression specificity and significancy for cancer. In an attempt to increase the targeted drug delivery to the α7 nAChR-overexpressing tumors, herein, α-conotoxin ImI, a disulfide-rich toxin with highly affinity for α7 nAChR, was modified on the PEG-DSPE micelles (ImI-PMs) for the first time. The DLS, TEM and HPLC detections showed the spherical nanoparticle morphology about 20 nm with negative charge and high drug encapsulation. The ligand modification did not induce significant differences. The immunofluorescence assay confirmed the expression level of α7 nAChR in MCF-7 cells. In vitro and in vivo experiments demonstrated that the α7 nAChR-targeted nanomedicines could deliver more specifically and faster into α7 nAChR-overexpressing MCF-7 cells. Furthermore, fluo-3/AM fluorescence imaging technique indicated that the increased specificity was attributed to the ligand-receptor interaction, and the inducitivity for intracellular Ca(2+) transient by ImI was still remained after modification. Moreover, paclitaxel, a clinical frequently-used anti-tumor drug for breast cancer, was loaded in ImI-modified nanomedicines to evaluate the targeting efficacy. Besides of exhibiting greater cytotoxicity and inducing more cell apoptosis in vitro, paclitaxel-loaded ImI-PMs displayed stronger anti-tumor efficacy in MCF-7 tumor-bearing nu/nu mice. Finally, the active targeting system showed low systemic toxicity and myelosuppression evidenced by less changes in body weight, white blood cells, neutrophilic granulocyte and platelet counts. In conclusion, α7 nAChR is also a promising target for anti-tumor drug delivery and in this case, α-conotoxin ImI-modified nanocarrier is a potential delivery system for targeting α7 nAChR-overexpressing tumors. PMID:25542793

  16. Chlorpyrifos and malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity.

    PubMed

    Richendrfer, Holly; Creton, Robbert

    2015-07-01

    Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. PMID:25983063

  17. Chlorpyrifos and Malathion have opposite effects on behaviors and brain size that are not correlated to changes in AChE activity

    PubMed Central

    Richendrfer, Holly; Creton, Robbert

    2015-01-01

    Organophosphates, a type of neurotoxicant pesticide, are used globally for the treatment of pests on croplands and are therefore found in a large number of conventional foods. These pesticides are harmful and potentially deadly if ingested or inhaled in large quantities by causing a significant reduction in acetylcholinesterase (AChE) activity in the central and peripheral nervous system. However, much less is known about the effects of exposure to small quantities of the pesticides on neural systems and behavior during development. In the current study we used zebrafish larvae in order to determine the effects of two of the most widely used organophosphates, chlorpyrifos and malathion, on zebrafish behavior and AChE activity. Embryos and larvae were exposed to the organophosphates during different time points in development and then tested at 5 days post-fertilization for behavioral, neurodevelopmental and AChE abnormalities. The results of the study indicate that chlorpyrifos and malathion cause opposing behaviors in the larvae such as swim speed (hypoactivity vs. hyperactivity) and rest. Additionally, the pesticides affect only certain behaviors, such as thigmotaxis, during specific time points in development that are unrelated to changes in AChE activity. Larvae treated with malathion but not chlorpyrifos also had significantly smaller forebrain and hindbrain regions compared to controls by 5 days post-fertilization. We conclude that exposure to very low concentrations of organophosphate pesticides during development cause abnormalities in behavior and brain size. PMID:25983063

  18. Acute and long-term exposure to chlorpyrifos induces cell death of basal forebrain cholinergic neurons through AChE variants alteration.

    PubMed

    del Pino, Javier; Moyano, Paula; Anadon, María José; García, José Manuel; Díaz, María Jesús; García, Jimena; Frejo, María Teresa

    2015-10-01

    Chlorpyrifos (CPF) is one of the most widely used organophosphates insecticides that has been reported to induce cognitive disorders both after acute and repeated administration similar to those induced in Alzheimer's disease (AD). However, the mechanisms through which it induces these effects are unknown. On the other hand, the cholinergic system, mainly basal forebrain cholinergic neurons, is involved in learning and memory regulation, and an alteration of cholinergic transmission or/and cholinergic cell loss could induce these effects. In this regard, it has been reported that CPF can affect cholinergic transmission, and alter AChE variants, which have been shown to be related with basal forebrain cholinergic neuronal loss. According to these data, we hypothesized that CPF could induce basal forebrain cholinergic neuronal loss through cholinergic transmission and AChE variants alteration. To prove this hypothesis, we evaluated in septal SN56 basal forebrain cholinergic neurons, the CPF toxic effects after 24h and 14 days exposure on neuronal viability and the cholinergic mechanisms related to it. This study shows that CPF impaired cholinergic transmission, induced AChE inhibition and, only after long-term exposure, increased CHT expression, which suggests that acetylcholine levels alteration could be mediated by these actions. Moreover, CPF induces, after acute and long-term exposure, cell death in cholinergic neurons in the basal forebrain and this effect is independent of AChE inhibition and acetylcholine alteration, but was mediated partially by AChE variants alteration. Our present results provide a new understanding of the mechanisms contributing to the harmful effects of CPF on neuronal function and viability, and the possible relevance of CPF in the pathogenesis of neurodegenerative diseases. PMID:26210949

  19. Structural Mechanisms of the Agrin-LRP4-MuSK Signaling Pathway in Neuromuscular Junction Differentiation

    PubMed Central

    Zong, Yinong; Jin, Rongsheng

    2015-01-01

    The neuromuscular junction (NMJ) is the most extensively studied model of neuronal synaptogenesis. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in the differentiation of NMJs. AChR clustering and postsynaptic differentiation is orchestrated by sophisticated interactions among three proteins: the neuron-secreted proteoglycan agrin, the co-receptor LRP4, and the muscle-specific receptor tyrosine kinase MuSK. LRP4 and MuSK act as scaffolds for multiple binding partners, resulting in a complex and dynamic network of interacting proteins that is required for AChR clustering. In this review, we discuss the structural basis for NMJ postsynaptic differentiation mediated by the agrin-LRP4-MuSK signaling pathway. PMID:23178848

  20. Structural mechanisms of the agrin-LRP4-MuSK signaling pathway in neuromuscular junction differentiation.

    PubMed

    Zong, Yinong; Jin, Rongsheng

    2013-09-01

    The neuromuscular junction (NMJ) is the most extensively studied model of neuronal synaptogenesis. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in the differentiation of NMJs. AChR clustering and postsynaptic differentiation is orchestrated by sophisticated interactions among three proteins: the neuron-secreted proteoglycan agrin, the co-receptor LRP4, and the muscle-specific receptor tyrosine kinase MuSK. LRP4 and MuSK act as scaffolds for multiple binding partners, resulting in a complex and dynamic network of interacting proteins that is required for AChR clustering. In this review, we discuss the structural basis for NMJ postsynaptic differentiation mediated by the agrin-LRP4-MuSK signaling pathway. PMID:23178848

  1. Phe362Tyr in AChE: A Major Factor Responsible for Azamethiphos Resistance in Lepeophtheirus salmonis in Norway.

    PubMed

    Kaur, Kiranpreet; Jansen, Peder Andreas; Aspehaug, Vidar Teis; Horsberg, Tor Einar

    2016-01-01

    Organophosphates (OP) are one of the major treatments used against the salmon louse (Lepeophtherius salmonis) in Norwegian salmonid aquaculture. The use of OP since the late 1970s has resulted in widespread resistant parasites. Recently, we reported a single mutation (Phe362Tyr) in acetylcholinesterase (AChE) as the major mechanism behind resistance in salmon louse towards OP. The present study was carried out to validate this mechanism at the field level. A total of 6658 salmon louse samples were enrolled from 56 different fish farms across the Norwegian coast, from Vest Agder in the south to Finnmark in the north. All the samples were genotyped using a TaqMan probe assay for the Phe362Tyr mutation. A strong association was observed between areas with frequent use of the OP (azamethiphos) and the Phe362Tyr mutation. This was confirmed at 15 sites where results from independently conducted bioassays and genotyping of parasites correlated well. Furthermore, genotyping of surviving and moribund parasites from six bioassay experiments demonstrated a highly significant negative correlation between the frequency of resistance alleles and the probability of dying when exposed to azamethiphos in a bioassay. Based on these observations, we could strongly conclude that the Phe362Tyr mutation is a major factor responsible for OP resistance in salmon louse on Norwegian fish farms. PMID:26882536

  2. Phe362Tyr in AChE: A Major Factor Responsible for Azamethiphos Resistance in Lepeophtheirus salmonis in Norway

    PubMed Central

    Kaur, Kiranpreet; Jansen, Peder Andreas; Aspehaug, Vidar Teis; Horsberg, Tor Einar

    2016-01-01

    Organophosphates (OP) are one of the major treatments used against the salmon louse (Lepeophtherius salmonis) in Norwegian salmonid aquaculture. The use of OP since the late 1970s has resulted in widespread resistant parasites. Recently, we reported a single mutation (Phe362Tyr) in acetylcholinesterase (AChE) as the major mechanism behind resistance in salmon louse towards OP. The present study was carried out to validate this mechanism at the field level. A total of 6658 salmon louse samples were enrolled from 56 different fish farms across the Norwegian coast, from Vest Agder in the south to Finnmark in the north. All the samples were genotyped using a TaqMan probe assay for the Phe362Tyr mutation. A strong association was observed between areas with frequent use of the OP (azamethiphos) and the Phe362Tyr mutation. This was confirmed at 15 sites where results from independently conducted bioassays and genotyping of parasites correlated well. Furthermore, genotyping of surviving and moribund parasites from six bioassay experiments demonstrated a highly significant negative correlation between the frequency of resistance alleles and the probability of dying when exposed to azamethiphos in a bioassay. Based on these observations, we could strongly conclude that the Phe362Tyr mutation is a major factor responsible for OP resistance in salmon louse on Norwegian fish farms. PMID:26882536

  3. Protective molecular mimicry in experimental myasthenia gravis.

    PubMed

    Im, Sin Hyeog; Barchan, Dora; Feferman, Tali; Raveh, Lily; Souroujon, Miriam C; Fuchs, Sara

    2002-05-01

    Protein databases were searched for microbial sequences that bear amino acid similarities with identified T- or B-cell epitopes within the human alpha-subunit of acetylcholine receptor (AChR). One peptide, derived from Haemophilus influenzae, exhibits 50% homology to an identified T-cell epitope of AChR alpha-subunit. This peptide was shown to have a protective effect in experimental autoimmune myasthenia gravis (EAMG). Pretreatment of rats with the mimicry peptide attenuated the induction and progression of EAMG. These effects were accompanied by a reduced T-cell response to AChR, diminished IL-2, IL-12, IFN-gamma and IL-4 levels, as well as decreased humoral response to self-AChR. PMID:12020961

  4. Pyridonepezils, new dual AChE inhibitors as potential drugs for the treatment of Alzheimer's disease: synthesis, biological assessment, and molecular modeling.

    PubMed

    Samadi, Abdelouahid; Estrada, Martín; Pérez, Concepción; Rodríguez-Franco, María Isabel; Iriepa, Isabel; Moraleda, Ignacio; Chioua, Mourad; Marco-Contelles, José

    2012-11-01

    The synthesis, biological assessment and molecular modeling of new pyridonepezils1-8, able to inhibit human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBuChE), are described. The new compounds have been designed as hybrids resulting from a conjunctive approach that combines the N-benzylpiperidine moiety, present in donepezil, and the 2-amino-6-chloropyridine heterocyclic ring system, connected by an appropriate polymethylene linker. Compounds 1-8 were prepared by reaction of 2-amino-6-chloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2-amino-6-chloropyridine-3,5-dicarbonitrile (14)] with 2-(1-benzylpiperidin-4-yl)alkylamines (9-12). The biological evaluation of molecules 1-8 showed that compounds 1-6 are potent AChE inhibitors, in the submicromolar, while compounds 7 and 8 are on the nanomolar range, the most potent, 2-amino-6-((3-(1-benzylpiperidin-4-yl)propyl)amino)pyridine-3,5-dicarbonitrile (7), showing a IC(50) (hAChE) = 9.4 ± 0.4 nM. Inhibitors 2-8 are permeable as determined in the PAMPA assay. Compared to donepezil, compound 7 is in the same range of inhibitory activity for hAChE, and 703-fold more selective for hAChE than for hBuChE. Molecular modeling investigation on pyridonepezil7 supports its dual AChE inhibitory profile, binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The theoretical ADME analysis of pyridonepezils1-8 has been carried out. Overall, compound 7, a potent and selective dual AChEI, can be considered as a candidate with potential impact for further pharmacological development in Alzheimer's therapy. PMID:23078965

  5. The role of the a7 subunit of the nicotinic acetylcholine receptor on motor coordination in mice treated with methyllcaconitine and anabasine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse effects of methyllycaconitine (MLA) have been attributed to competitive antagonism of nicotinic acetylcholine receptors (nAChR). Research has indicated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice with more a7 nAChR requi...

  6. Innate immunity in myasthenia gravis thymus: pathogenic effects of Toll-like receptor 4 signaling on autoimmunity.

    PubMed

    Cordiglieri, Chiara; Marolda, Roberta; Franzi, Sara; Cappelletti, Cristina; Giardina, Carmelo; Motta, Teresio; Baggi, Fulvio; Bernasconi, Pia; Mantegazza, Renato; Cavalcante, Paola

    2014-08-01

    The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naïve CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses. PMID:24397961

  7. Vaccines against myasthenia gravis

    PubMed Central

    Berrih-Aknin, Sonia; Fuchs, Sara; Souroujon, Miriam C

    2007-01-01

    Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies to nicotinic acetylcholine receptor (AChR) interfering with the neuromuscular transmission. Experimental autoimmune MG serves as an excellent animal model to study possible therapeutic modalities for MG. This review will focus on the different ways to turn off the autoimmune response to AChR, which results in suppression of myasthenia. This paper will describe the use of fragments or peptides derived from the AChR, antigen-presenting cells and anti-T cell receptor antibodies, and will discuss the underlying mechanisms of action. Finally, the authors propose new promising therapeutic prospects, including treatment based on the modulation of regulatory T cells, which have recently been found to be functionally defective in MG patients. PMID:16018742

  8. Autoantibody profile and clinical characteristics in a cohort of Chinese adult myasthenia gravis patients.

    PubMed

    Hong, Yu; Li, Hai-Feng; Skeie, Geir Olve; Romi, Fredrik; Hao, Hong-Jun; Zhang, Xu; Gao, Xiang; Owe, Jone Furlund; Gilhus, Nils Erik

    2016-09-15

    Myasthenia gravis (MG) is an autoimmune disorder with heterogeneity. Antibodies against acetylcholine receptor (AChR), muscle-specific kinase (MuSK), titin and ryanodine receptor (RyR) were examined in 437 adult Chinese MG patients. The AChR, MuSK, titin and RyR antibodies were found in 82.2%, 2.3%, 28.4% and 23.8% of all patients. Autoantibody profiles vary among different MG subgroups. Thymoma MG patients had high frequencies of AChR (99.2%), titin (50.8%) and RyR antibodies (46.9%). The titin and RyR antibodies also showed high frequencies in late onset patients (54.4% and 33.3%, respectively). These two antibodies may indicate an underlying thymoma when combined. The patients with titin and RyR antibodies tend to have more severe disease and worse outcome, and may need more active immunosuppressive treatment. PMID:27609275

  9. Extracts and constituents of Leontopodium alpinum enhance cholinergic transmission: Brain ACh increasing and memory improving properties

    PubMed Central

    Hornick, Ariane; Schwaiger, Stefan; Rollinger, Judith M.; Vo, Nguyen Phung; Prast, Helmut; Stuppner, Hermann

    2012-01-01

    Leontopodium alpinum (‘Edelweiss’) was phytochemically investigated for constituents that might enhance cholinergic neurotransmission. The potency to increase synaptic availability of acetylcholine (ACh) in rat brain served as key property for the bioguided isolation of cholinergically active compounds using different chromatographic techniques. The dichlormethane (DCM) extract of the root, fractions and isolated constituents were injected i.c.v. and the effect on brain ACh was detected via the push–pull technique. The DCM extract enhanced extracellular ACh concentration in rat brain and inhibited acetylcholinesterase (AChE) in vitro. The extracellular level of brain ACh was significantly increased by the isolated sesquiterpenes, isocomene and 14-acetoxyisocomene, while silphiperfolene acetate and silphinene caused a small increasing tendency. Only silphiperfolene acetate showed in vitro AChE inhibitory activity, thus suggesting the other sesquiterpenes to stimulate cholinergic transmission by an alternative mechanism of action. Isocomene was further investigated with behavioural tasks in mice. It restored object recognition in scopolamine-impaired mice and showed nootropic effects in the T-maze alternation task in normal and scopolamine-treated mice. Additionally, this sesquiterpene reduced locomotor activity of untreated mice in the open field task, while the activity induced by scopolamine was abolished. The enhancement of synaptic availability of ACh, the promotion of alternation, and the amelioration of scopolamine-induced deficit are in accordance with a substance that amplifies cholinergic transmission. Whether the mechanism of action is inhibition of AChE or another pro-cholinergic property remains to be elucidated. Taken together, isocomene and related constituents of L. alpinum deserve further interest as potential antidementia agents in brain diseases associated with cholinergic deficits. PMID:18541221

  10. Roles of nicotinic acetylcholine receptor β subunits in function of human α4-containing nicotinic receptors

    PubMed Central

    Wu, Jie; Liu, Qiang; Yu, Kewei; Hu, Jun; Kuo, Yen-Ping; Segerberg, Marsha; St John, Paul A; Lukas, Ronald J

    2006-01-01

    Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*-nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2- and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2- and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*-nAChR with distinctive physiological and pharmacological features. Diversity in α4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence. PMID:16825297

  11. Evaluation of Z-(R,R)-IQNP for the potential imaging of m2 mAChR rich regions of the brain and heart.

    PubMed

    McPherson, D W; Greenbaum, M; Luo, H; Beets, A L; Knapp, F F

    2000-01-01

    Alterations in the function or density of the m2 muscarinic (mAChR) subtype have been postulated to play an important role in various dementias such as Alzheimer's disease. The ability to image and quantify the m2 mAChR subtype is of importance for a better understanding of the m2 subtype function in various dementias. Z-(R)-1-Azabicyclo[2.2.2]oct-3-y (R)-alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (Z-(R,R)-IQNP) has demonstrated significant uptake in cerebral regions that contain a high concentration of m2 mAChR subtype in addition to heart tissue. The present study was undertaken to determine if the uptake of Z-(R,R)-IQNP in these regions is a receptor mediated process and to identify the radiospecies responsible for binding at the receptor site. A blocking study demonstrated cerebral and cardiac levels of activity were significantly reduced by pretreatment (2-3 mg/kg) of (R)-3-quinuclidinyl benzilate, dexetimide and scopolamine, established muscarinic antagonists. A direct comparison of the cerebral and cardiac uptake of [I-125]-Z-(R,R)-IQNP and [I-131]-E-(R,R)-IQNP (high uptake in ml, m4 rich mAChR cerebral regions) demonstrated Z-(R,R)-IQNP localized to a higher degree in cerebral and cardiac regions containing a high concentration of the m2 mAChR subtype as directly compared to E-(R,R)-IQNP. In addition, a study utilizing [I-123]-Z-(R,R)-IQNP, [I-131]-iododexetimide and [I-125]-R-3-quinuclidinyl S-4-iodobenzilate, Z-(R,R)-IQNP demonstrated significantly higher uptake and longer residence time in those regions which contain a high concentration of the m2 receptor subtype. Folch extraction of global brain and heart tissue at various times post injection of [I-125]-Z-(R,R)-IQNP demonstrated that approximately 80% of the activity was extracted in the lipid soluble fraction and identified as the parent ligand by TLC and HPLC analysis. These results demonstrate Z-(R,R)-IQNP has significant uptake, long residence time and high stability in

  12. Highly Selective and Sensitive Detection of Acetylcholine Using Receptor-Modified Single-Walled Carbon Nanotube Sensors

    NASA Astrophysics Data System (ADS)

    Xu, Shihong; Kim, Byeongju; Song, Hyun Seok; Jin, Hye Jun; Park, Eun Jin; Lee, Sang Hun; Lee, Byung Yang; Park, Tai Hyun; Hong, Seunghun

    2015-03-01

    Acetylcholine (ACh) is a neurotransmitter in a human central nervous system and is related to various neural functions such as memory, learning and muscle contractions. Dysfunctional ACh regulations in a brain can induce several neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease and myasthenia gravis. In researching such diseases, it is important to measure the concentration of ACh in the extracellular fluid of the brain. Herein, we developed a highly sensitive and selective ACh sensor based on single-walled carbon nanotube-field effect transistors (swCNT-FETs). In our work, M1 mAChR protein, an ACh receptor, was expressed in E.coli and coated on swCNT-FETs with lipid membranes. Here, the binding of ACh onto the receptors could be detected by monitoring the change of electrical currents in the underlying swCNT-FETs, allowing the real-time detection of ACh at a 100 pM concentration. Furthermore, our sensor could selectively detect ACh from other neurotransmitters. This is the first report of the real-time sensing of ACh utilizing specific binding between the ACh and M1 mAChR, and it may lead to breakthroughs in various biomedical applications such as drug screening and disease diagnosis.

  13. Novel potent pyridoxine-based inhibitors of AChE and BChE, structural analogs of pyridostigmine, with improved in vivo safety profile.

    PubMed

    Strelnik, Alexey D; Petukhov, Alexey S; Zueva, Irina V; Zobov, Vladimir V; Petrov, Konstantin A; Nikolsky, Evgeny E; Balakin, Konstantin V; Bachurin, Sergey O; Shtyrlin, Yurii G

    2016-08-15

    We report a novel class of carbamate-type ChE inhibitors, structural analogs of pyridostigmine. A small library of congeneric pyridoxine-based compounds was designed, synthesized and evaluated for AChE and BChE enzymes inhibition in vitro. The most active compounds have potent enzyme inhibiting activity with IC50 values in the range of 0.46-2.1μM (for AChE) and 0.59-8.1μM (for BChE), with moderate selectivity for AChE comparable with that of pyridostigmine and neostigmine. Acute toxicity studies using mice models demonstrated excellent safety profile of the obtained compounds with LD50 in the range of 22-326mg/kg, while pyridostigmine and neostigmine are much more toxic (LD50 3.3 and 0.51mg/kg, respectively). The obtained results pave the way to design of novel potent and safe cholinesterase inhibitors for symptomatic treatment of neuromuscular disorders. PMID:27377327

  14. Design of multi-target compounds as AChE, BACE1, and amyloid-β(1-42) oligomerization inhibitors: in silico and in vitro studies.

    PubMed

    Hernández-Rodríguez, Maricarmen; Correa-Basurto, José; Martínez-Ramos, Federico; Padilla-Martínez, Itzia Irene; Benítez-Cardoza, Claudia G; Mera-Jiménez, Elvia; Rosales-Hernández, Martha Cecilia

    2014-01-01

    Despite great efforts to develop new therapeutic strategies against Alzheimer's disease (AD), the acetylcholinesterase inhibitors (AChEIs): donepezil, rivastigmine, and galantamine, have been used only as a palliative therapeutic approach. However, the pathogenesis of AD includes several factors such as cholinergic hypothesis, amyloid-β (Aβ) aggregation, and oxidative stress. For this reason, the design of compounds that target the genesis and progression of AD could offer a therapeutic benefit. We have designed a set of compounds (M-1 to M-5) with pharmacophore moieties to inhibit the release, aggregation, or toxicity of Aβ, act as AChEIs and have antioxidant properties. Once the compounds were designed, we analyzed their physicochemical parameters and performed docking studies to determine their affinity values for AChE, β-site amyloid-protein precursor cleaving enzyme 1 (BACE1), and the Aβ monomer. The best ligands, M-1 and M-4, were then synthesized, chemically characterized, and evaluated in vitro. The in vitro studies showed that these compounds inhibit AChE (M-1 Ki = 0.12 and M-4 Ki = 0.17 μM) and BACE1 (M-1 IC50 = 15.1 and M-4 IC50 = 15.4 nM). They also inhibit Aβ oligomerization and exhibit antioxidant activity. In addition, these compounds showed low cytotoxicity in microglial cells. For these reasons, they are promising for future use as drugs in AD mice transgenic models. PMID:24762947

  15. Marine Macrocyclic Imines, Pinnatoxins A and G: Structural Determinants and Functional Properties to Distinguish Neuronal α7 from Muscle α1(2)βγδ nAChRs.

    PubMed

    Bourne, Yves; Sulzenbacher, Gerlind; Radić, Zoran; Aráoz, Rómulo; Reynaud, Morgane; Benoit, Evelyne; Zakarian, Armen; Servent, Denis; Molgó, Jordi; Taylor, Palmer; Marchot, Pascale

    2015-06-01

    Pinnatoxins are macrocyclic imine phycotoxins associated with algal blooms and shellfish toxicity. Functional analysis of pinnatoxin A and pinnatoxin G by binding and voltage-clamp electrophysiology on membrane-embedded neuronal α7, α4β2, α3β2, and muscle-type α12βγδ nicotinic acetylcholine receptors (nAChRs) reveals high-affinity binding and potent antagonism for the α7 and α12βγδ subtypes. The toxins also bind to the nAChR surrogate, acetylcholine-binding protein (AChBP), with low Kd values reflecting slow dissociation. Crystal structures of pinnatoxin-AChBP complexes (1.9-2.2 Å resolution) show the multiple anchoring points of the hydrophobic portion, the cyclic imine, and the substituted bis-spiroketal and cyclohexene ring systems of the pinnatoxins that dictate tight binding between the opposing loops C and F at the receptor subunit interface, as observed for the 13-desmethyl-spirolide C and gymnodimine A congeners. Uniquely, however, the bulky bridged EF-ketal ring specific to the pinnatoxins extends radially from the interfacial-binding pocket to interact with the sequence-variable loop F and govern nAChR subtype selectivity and central neurotoxicity. PMID:26004441

  16. Nicotinic acetylcholine receptor expression in human airway correlates with lung function.

    PubMed

    Lam, David Chi-Leung; Luo, Susan Yang; Fu, Kin-Hang; Lui, Macy Mei-Sze; Chan, Koon-Ho; Wistuba, Ignacio Ivans; Gao, Boning; Tsao, Sai-Wah; Ip, Mary Sau-Man; Minna, John Dorrance

    2016-02-01

    Nicotine and its derivatives, by binding to nicotinic acetylcholine receptors (nAChRs) on bronchial epithelial cells, can regulate cellular signaling and inflammatory processes. Delineation of nAChR subtypes and their responses to nicotine stimulation in bronchial epithelium may provide information for therapeutic targeting in smoking-related inflammation in the airway. Expression of nAChR subunit genes in 60 bronchial epithelial biopsies and immunohistochemical staining for the subcellular locations of nAChR subunit expression were evaluated. Seven human bronchial epithelial cell lines (HBECs) were exposed to nicotine in vitro for their response in nAChR subunit gene expression to nicotine exposure and removal. The relative normalized amount of expression of nAChR α4, α5, and α7 and immunohistochemical staining intensity of nAChR α4, α5, and β3 expression showed significant correlation with lung function parameters. Nicotine stimulation in HBECs resulted in transient increase in the levels of nAChR α5 and α6 but more sustained increase in nAChR α7 expression. nAChR expression in bronchial epithelium was found to correlate with lung function. Nicotine exposure in HBECs resulted in both short and longer term responses in nAChR subunit gene expression. These results gave insight into the potential of targeting nAChRs for therapy in smoking-related inflammation in the airway. PMID:26608528

  17. Coantagonism of Glutamate Receptors and Nicotinic Acetylcholinergic Receptors Disrupts Fear Conditioning and Latent Inhibition of Fear Conditioning

    ERIC Educational Resources Information Center

    Gould, Thomas J.; Lewis, Michael C.

    2005-01-01

    The present study investigated the hypothesis that both nicotinic acetylcholinergic receptors (nAChRs) and glutamate receptors ([alpha]-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) and N-methyl-D-aspartate glutamate receptors (NMDARs)) are involved in fear conditioning, and may modulate similar processes. The effects of the…

  18. Acetylcholine receptor and behavioral deficits in mice lacking apolipoprotein E

    PubMed Central

    Siegel, Jessica A; Benice, Theodore S; Van Meer, Peter; Park, Byung S; Raber, Jacob

    2011-01-01

    Apolipoprotein E (apoE) is involved in the risk to develop sporadic Alzheimer’s disease (AD). Since impaired central acetylcholine (ACh) function is a hallmark of AD, apoE may influence ACh function by modulating muscarinic ACh receptors (mAChRs). To test this hypothesis, mAChR binding was measured in mice lacking apoE and wild type C57BL/6J mice. Mice were also tested on the pre-pulse inhibition, delay eyeblink classical conditioning, and 5-choice serial reaction time tasks, which are all modulated by ACh transmission. Mice were also given scopolamine to challenge central mAChR function. Compared to wild type mice, mice lacking apoE had reduced number of cortical and hippocampal mAChRs. Scopolamine had a small effect on delay eyeblink classical conditioning in wild type mice but a large effect in mice lacking apoE. Mice lacking apoE were also unable to acquire performance on the 5-choice serial reaction time task. These results support a role for apoE in ACh function and suggest that modulation of cortical and hippocampal mAChRs might contribute to genotype differences in scopolamine sensitivity and task acquisition. Impaired apoE functioning may result in cholinergic deficits that contribute to the cognitive impairments seen in AD. PMID:19178986

  19. 31 CFR 363.38 - What happens if my financial institution returns an ACH debit?

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 2 2010-07-01 2010-07-01 false What happens if my financial... TreasuryDirect § 363.38 What happens if my financial institution returns an ACH debit? If your designated...Direct ® account. If the ACH return occurs after the security has been redeemed, transferred, or...

  20. 31 CFR 363.38 - What happens if my financial institution returns an ACH debit?

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 2 2011-07-01 2011-07-01 false What happens if my financial... TreasuryDirect § 363.38 What happens if my financial institution returns an ACH debit? If your designated...Direct ® account. If the ACH return occurs after the security has been redeemed, transferred, or...

  1. THE ACHES THAT TAKE YOUR BREATH (AND TEARS) AWAY.

    PubMed

    Becerril, J; Gonzales, H; Saketkoo, L A

    2015-01-01

    An 80-year-old man presented with a complaint of three months of fatigue and aching of his shoulders and hips, as well as pain, swelling, and stiffness in bilateral fingers that was worse in the morning but improved with movement. Associated symptoms included worsening dry mouth and eyes, dysphagia, exertional dyspnea, and right foot drop. Physical exam was significant for edematous and tender bilateral proximal interphalangeal joints, metacarpophalangeal joints and wrists with decreased grip, extension and flexion, as well as bilateral pulmonary crackles. Laboratory analysis revealed Anti-Ro (SSA) and Anti-La (SSB) positivity with elevated erythrocyte sedimentation rate (70mm/hr) and C-reactive peptide (13mg/L). Pulmonary function testing was notable for a forced vital capacity (FVC) of 64% and carbon monoxide diffusing capacity (DLCO) of 44%. High resolution chest computed tomography demonstrated fibrotic changes consistent with nonspecific interstitial pneumonitis. The patient was started on mycophenolate mofetil, hydroxychloroquine, and prednisone for Sjögren's syndrome (SjS). Symptoms improved and repeat FVC revealed a 20 percent improvement, however subsequent tapering of prednisone resulted in worsening dyspnea and increase of FVC to 60 prcent. Prednisone was restarted and rituximab 2g divided in two doses was administered with overall symptom improvement. Symptoms and FVC continued to wax and wane over the following 18 months requiring re-dosing of rituximab with most recent FVC improved to 71 percent and DLCO 41 percent. PMID:27159479

  2. Pharmacological stress is required for the anti-alcohol effect of the α3β4* nAChR partial agonist AT-1001

    PubMed Central

    Cippitelli, Andrea; Brunori, Gloria; Gaiolini, Kelly A.; Zaveri, Nurulain T.; Toll, Lawrence

    2015-01-01

    Alcohol and nicotine are often taken together. The mechanisms underlying this frequent co-abuse are not well known. Genetic and pharmacological evidence suggests that the nicotinic acetylcholine receptors (nAChRs) containing the α3 and β4 subunits play a role in alcohol as well as nicotine addiction. AT-1001 is a high affinity α3β4 nAChR partial agonist recently found to block nicotine self-administration and relapse-like behavior in rats. Here, to study the involvement of α3β4 nAChRs in the mechanisms that regulate alcohol abuse we evaluated the effects of AT-1001 on alcohol taking and seeking in Sprague-Dawley rats. AT-1001 reduced operant alcohol self-administration at the highest dose examined (3.0 mg/kg), an effect also observed for food self-administration. A dose of 1.5 mg/kg AT-1001, which had no effect on alcohol or food self-administration, essentially eliminated reinstatement of alcohol seeking induced by yohimbine (0.625 mg/kg) whereas, reinstatement induced by alcohol-associated cues was not altered, nor did AT-1001 induce reinstatement of extinguished self-administration on its own. Finally, AT-1001 showed an anxiolytic activity when measured in the presence or absence of yohimbine stress in the elevated plus maze paradigm. Together, these observations do not support a specific involvement of the α3β4 nAChR in mediating alcohol reward or cue-induced relapse to alcohol seeking but rather indicate that the α3β4 nAChR partial agonism may constitute an attractive approach for treating alcohol use disorders exacerbated by elevated stress response. PMID:25689019

  3. Pharmacological stress is required for the anti-alcohol effect of the α3β4* nAChR partial agonist AT-1001.

    PubMed

    Cippitelli, Andrea; Brunori, Gloria; Gaiolini, Kelly A; Zaveri, Nurulain T; Toll, Lawrence

    2015-06-01

    Alcohol and nicotine are often taken together. The mechanisms underlying this frequent co-abuse are not well known. Genetic and pharmacological evidence suggests that the nicotinic acetylcholine receptors (nAChRs) containing the α3 and β4 subunits play a role in alcohol as well as nicotine addiction. AT-1001 is a high affinity α3β4 nAChR partial agonist recently found to block nicotine self-administration and relapse-like behavior in rats. Here, to study the involvement of α3β4 nAChRs in the mechanisms that regulate alcohol abuse we evaluated the effects of AT-1001 on alcohol taking and seeking in Sprague-Dawley rats. AT-1001 reduced operant alcohol self-administration at the highest dose examined (3.0 mg/kg), an effect also observed for food self-administration. A dose of 1.5 mg/kg AT-1001, which had no effect on alcohol or food self-administration, essentially eliminated reinstatement of alcohol seeking induced by yohimbine (0.625 mg/kg) whereas, reinstatement induced by alcohol-associated cues was not altered, nor did AT-1001 induce reinstatement of extinguished self-administration on its own. Finally, AT-1001 showed an anxiolytic activity when measured in the presence or absence of yohimbine stress in the elevated plus maze paradigm. Together, these observations do not support a specific involvement of the α3β4 nAChR in mediating alcohol reward or cue-induced relapse to alcohol seeking but rather indicate that the α3β4 nAChR partial agonism may constitute an attractive approach for treating alcohol use disorders exacerbated by elevated stress response. PMID:25689019

  4. Anniston community health survey: Follow-up and dioxin analyses (ACHS-II)--methods.

    PubMed

    Birnbaum, Linda S; Dutton, N D; Cusack, C; Mennemeyer, S T; Pavuk, M

    2016-02-01

    High serum concentrations of polychlorinated biphenyls (PCBs) have been reported previously among residents of Anniston, Alabama, where a PCB production facility was located in the past. As the second of two cross-sectional studies of these Anniston residents, the Anniston Community Health Survey: Follow-Up and Dioxin Analyses (ACHS-II) will yield repeated measurements to be used to evaluate changes over time in ortho-PCB concentrations and selected health indicators in study participants. Dioxins, non-ortho PCBs, other chemicals, heavy metals, and a variety of additional clinical tests not previously measured in the original ACHS cohort will be examined in ACHS-II. The follow-up study also incorporates a questionnaire with extended sections on diet and occupational history for a more comprehensive assessment of possible exposure sources. Data collection for ACHS-II from 359 eligible participants took place in 2014, 7 to 9 years after ACHS. PMID:25982988

  5. Central loop of non-conventional toxin WTX from Naja kaouthia is important for interaction with nicotinic acetylcholine receptors.

    PubMed

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Shenkarev, Zakhar O; Kasheverov, Igor E; Chugunov, Anton O; Kulbatskii, Dmitrii S; Myshkin, Mikhail Yu; Utkin, Yuri N; Efremov, Roman G; Tsetlin, Victor I; Arseniev, Alexander S; Kirpichnikov, Mikhail P; Dolgikh, Dmitry A

    2016-09-01

    'Three-finger' toxin WTX from Naja kaouthia interacts with nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs). Mutagenesis and competition experiments with (125)I-α-bungarotoxin revealed that Arg31 and Arg32 residues from the WTX loop II are important for binding to Torpedo californica and human α7 nAChRs. Computer modeling suggested that loop II occupies the orthosteric binding site at α7 nAChR. The similar toxin interface was previously described as a major determinant of allosteric interactions with mAChRs. PMID:27343701

  6. Molecular Mechanisms of Cross-inhibition Between Nicotinic Acetylcholine Receptors and P2X Receptors in Myenteric Neurons and HEK-293 cells

    PubMed Central

    Decker, Dima A.; Galligan, James J.

    2010-01-01

    Background P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit-containing nAChRs. Methods The function of P2X2 and α3β4 nAChRs expressed by HEK-293 cells and guinea pig ileum myenteric neurons in culture was studied using whole-cell patch clamp techniques. Results In HEK-293 cells expressing α3β4 nAChRs and P2X2 receptors, co-application of ATP and ACh caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross-inhibition. Cross-inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs and a C-terminal tail truncated P2X2 receptor (P2X2TR)(P >0.05, n = 8). Intracellular application of the C-terminal tail of the P2X2 receptor blocked nAChR-P2X receptor cross-inhibition in HEK-293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK-293 cells expressing both receptors. Constitutive inhibition did not occur in HEK-293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μM), but not high (≥100 μM) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co-expression with α3β4 nAChRs. Conclusions The C-terminal tail of P2X2 receptors mediates cross-inhibition between α3β4 nAChR-P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co-transmitters. PMID:20426799

  7. Toxicity and mAChRs binding activity of Cassiopea xamachana venom from Puerto Rican coasts.

    PubMed

    Radwan, Faisal F Y; Román, Laura G; Baksi, Krishna; Burnett, Joseph W

    2005-01-01

    A separation of toxic components from the upside down jellyfish Cassiopea xamachana (Cx) was carried out to study their cytotoxic effects and examine whether these effects are combined with a binding activity to cell membrane receptors. Nematocysts containing toxins were isolated from the autolysed tentacles, ruptured by sonication, and the crude venom (CxTX) was separated from the pellets by ultracentrifugation. For identifying its bioactive components, CxTX was fractionated by gel filtration chromatography into six fractions (named fraction I-VI). The toxicity of CxTX and fractions was tested on mice; however, the hemolytic activity was tested on saline washed human erythrocytes. The LD50 of CxTX was 0.75 microg/g of mouse body and for fraction III, IV and VI were 0.28, 0.25 and 0.12 microg/g, respectively. Fractions I, II and V were not lethal at doses equivalent to LD50 1 microg/g. The hemolytic and phospholipase A2 (PLA2) activities of most fractions were well correlated with their mice toxicity. However, fraction VI, which contains the low molecular mass protein components (< or =10 kDa), has shown no PLA2 activity but highest toxicity to mice, highest hemolytic activity, and bound significantly to the acetylcholine muscarinic receptors (mAChRs) isolated from rat brain. The results suggested that fraction VI contains proteinaceous components contributing to most of cytolysis as well as membrane binding events. Meanwhile, fraction IV has shown high PLA2 that may contribute to the venom lethality and paralytic effects. PMID:15581689

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

    PubMed

    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

  9. [Recent advance in research for myasthenia gravis, in relation to various antibodies affecting synaptic structure and function].

    PubMed

    Takamori, Masaharu

    2009-11-01

    Autoantibodies impair acetylcholine receptor (AChR) in myasthenia gravis (MG) and P/Q-type voltage-gated calcium channel (VGCC) in Lambert-Eaton myasthenic syndrome (LEMS). (1) Some of MG and LEMS patients are "seronegative" for respective antibodies or modified by antibodies that recognize other proteins than AChR and VGCC such as MuSK, AChR allosteric site, membrane Na+ channel and ryanodine receptor-1 (RyR1) in MG, and synaptotagmin-1 in LEMS. (2) Autoimmune responses affect the proteins participating in the mechanisms to compensate for synaptic disorders on the basis of presynaptic Ca2+ homeostasis provided by VGCC and non-VGCC (receptor-operated TRPCs): they act as enhancers of Ca(2+) -mediated ACh release via phospholipase C signaling pathways including M1-type presynaptic muscarinic AChR, neurotrophin receptor (TrkB), and fast-mode of synaptic vesicle recycling. (3) The pathophysiology contributive to contractile fatigue in MG includes RyR1 and also TRPC3. The TRPC3 also forms a complex with STIM1 and Orail to make up for Ca2+ after sarcoplasmic Ca2+ release. The prevalent detection of anti-TRPC3 antibodies in MG with thymoma could affect muscle contractile machineries in addition to anti-RyR1-induced affection. (4) When one faces "seronegative" MG, one should be cautious to conformation-specific antibodies and also congenital myasthenic syndromes. PMID:20030211

  10. Ni nanoparticle catalyzed growth of MWCNTs on Cu NPs @ a-C:H substrate

    NASA Astrophysics Data System (ADS)

    Ghodselahi, T.; Solaymani, S.; Akbarzadeh Pasha, M.; Vesaghi, M. A.

    2012-11-01

    NiCu NPs @ a-C:H thin films with different Cu content were prepared by co-deposition by RF-sputtering and RF-plasma enhanced chemical vapor deposition (RF-PECVD) from acetylene gas and Cu and Ni targets. The prepared samples were used as catalysts for growing multi-wall carbon nanotubes (MWCNTs) from liquid petroleum gas (LPG) at 825 °C by thermal chemical vapor deposition (TCVD). By addition of Cu NPs @ a-C:H thin layer as substrate for Ni NPs catalyst, the density of the grown CNTs is greatly enhanced in comparison to bare Si substrate. Furthermore the average diameter of the grown CNTs decreases by decreasing of Cu content of Cu NPs @ a-C:H thin layer. However Cu NPs @ a-C:H by itself has no catalytic property in MWCNTs growth. Morphology and electrical and optical properties of Cu NPs @ a-C:H thin layer is affected by Cu content and each of them is effective parameter on growth of MWCNTs based on Ni NPs catalyst. Moreover, adding of a low amount of Ni NPs doesn't vary optical, electrical and morphology properties of Cu NPs @ a-C:H thin layer but it has a profound effect on its catalytic activity. Finally the density and diameter of MWCNTs can be optimized by selection of the Cu NPs @ a-C:H thin layer as substrate of Ni NPs.

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

    PubMed Central

    Murray, Teresa A; Liu, Qiang; Whiteaker, Paul; Wu, Jie; Lukas, Ronald J

    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 α7 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 α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy. Results: Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion: nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion. PMID:19498423

  12. Myasthenia gravis and congenital myasthenic syndromes in dogs and cats: A history and mini-review.

    PubMed

    Shelton, G Diane

    2016-06-01

    Myasthenia gravis (MG) is a disorder of neuromuscular transmission in which muscle weakness results from an autoantibody mediated depletion of acetylcholine receptors (AChRs) at the neuromuscular junction. Myasthenia gravis occurs spontaneously in dogs and cats, and as in human MG, an autoimmune response against nicotinic AChRs has been demonstrated and autoantibodies against AChRs implicated in the pathogenesis. While both species are affected with MG, there are distinct differences in clinical presentations and frequency of spontaneous remission. Congenital myasthenic syndromes (CMSs) are hereditary disorders of neuromuscular transmission resulting in structural or functional defects of the neuromuscular junction. The clinical presentation and pathogenesis of a CMS in Jack Russell terriers was first described in the 1970's and 1980s and has since been reported in a few other breeds. Mutations have been reported in CHRNE, COLQ and CHAT in canine CMS. A form of COLQ deficient CMS has recently been reported in cats. PMID:27080328

  13. Synthesis, pharmacological assessment, and molecular modeling of 6-chloro-pyridonepezils: new dual AChE inhibitors as potential drugs for the treatment of Alzheimer's disease.

    PubMed

    Samadi, Abdelouahid; de la Fuente Revenga, Mario; Pérez, Concepción; Iriepa, Isabel; Moraleda, Ignacio; Rodríguez-Franco, María Isabel; Marco-Contelles, José

    2013-09-01

    6-Chloro-pyridonepezils are chloropyridine-donepezil hybrids designed by combining the N-benzylpiperidine moiety present in donepezil with the 2-chloropyridine-3,5-dicarbonitrile heterocyclic ring system, both connected by an appropriate polymethylene linker. 6-Chloro-pyridonepezils1-8 were prepared by reaction of 2,6-dichloro-4-phenylpyridine-3,5-dicarbonitrile (13) [or 2,6-dichloropyridine-3,5-dicarbonitrile (14)] with suitable 2-(1-benzylpiperidin-4-yl)alkylamines (9-12). The biological evaluation showed that these new compounds are cholinesterase inhibitors, in the submicromolar range, one of them (6) being a potent hBuChE inhibitor (IC50 = 0.47 ± 0.08 μM). 6-Chloro-pyridonepezils4, 7 and 8 are potent hAChE inhibitors showing IC50 in the 0.013-0.054 μM range. Particularly, 6-chloro-pyridonepezil8 is 625-fold more selective for hAChE than for hBuChE and compared to donepezil is equipotent for the inhibition of hAChE. Molecular modeling investigation on 6-chloro-pyridonepezils4, 6-8 supports its dual AChE inhibitory profile, by binding simultaneously at the catalytic active and at peripheral anionic sites of the enzyme. The in vitro Blood Brain Barrier (BBB) and theoretical ADME analysis of 6-chloro-pyridonepezils1-8 have been carried out. Overall, compound 8, is a permeable potent and selective dual AChEI that can be considered as a good candidate with potential impact for further pharmacological development in Alzheimer's therapy. PMID:23838422

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

    PubMed

    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

  15. New quinoline derivatives as nicotinic receptor modulators.

    PubMed

    Manetti, Dina; Bellucci, Cristina; Dei, Silvia; Teodori, Elisabetta; Varani, Katia; Spirova, Ekaterina; Kudryavtsev, Denis; Shelukhina, Irina; Tsetlin, Victor; Romanelli, Maria Novella

    2016-03-01

    As a continuation of previous work on quinoline derivatives, which showed some preference (2-3 times) for the α7 with respect to α4β2 acetylcholine nicotinic receptors (nAChRs), we synthesized a series of novel azabicyclic or diazabicyclic compounds carrying a quinoline or isoquinoline ring, with the aim of searching for more selective α7 nAChR compounds. Radioligand binding studies on α7* and α4β2* nAChRs (rat brain homogenate) revealed one compound (7) with a 2-fold higher affinity for the α4β2*-subtype, and four compounds (11, 13, 14 and 16) with at least 3-fold higher affinity for α7* nAChR. The most promising was 11, showing Ki∼100 nM and over 10-fold selectivity for α7* nAChR. Compounds 7, 11, 13 and 16 at 50 μM suppressed ion currents induced in the rat α4β2 nAChR and the chimeric nAChR composed of the ligand-binding domain of the chick α7 and transmembrane domain of the α1 glycine receptor, expressed in Xenopus oocytes. Calcium imaging experiments on the human α7 nAChR expressed in the Neuro2a cells and potentiated by PNU-120596 confirmed the antagonistic activity for 7; on the contrary, 11, 13 and 16 were agonists with the EC50 values in the range of 1.0-1.6 μM. Thus, the introduced modifications allowed us to enhance the selectivity of quinolines towards α7 nAChR and to get novel compounds with agonistic activity. PMID:26840365

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

    PubMed

    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

  17. Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime in Aplysia.

    PubMed

    Fossier, P; Baux, G; Poulain, B; Tauc, L

    1990-09-01

    1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation. PMID:2253262

  18. α7 Nicotinic Acetylcholine Receptors Occur at Postsynaptic Densities of AMPA Receptor-Positive and -Negative Excitatory Synapses in Rat Sensory Cortex

    PubMed Central

    Levy, Robert B.; Aoki, Chiye

    2010-01-01

    NMDA receptor (NMDAR) activation requires concurrent membrane depolarization, and glutamatergic synapses lacking AMPA receptors (AMPARs) are often considered “silent” in the absence of another source of membrane depolarization. During the second postnatal week, NMDA currents can be enhanced in rat auditory cortex through activation of the α7 nicotinic acetylcholine receptor (α7nAChR). Electrophysiological results support a mainly presynaptic role for α7nAChR at these synapses. However, immunocytochemical evidence that α7nAChR is prevalent at postsynaptic sites of glutamatergic synapses in hippocampus and neocortex, along with emerging electrophysiological evidence for postsynaptic nicotinic currents in neocortex and hippocampus, has prompted speculation that α7nAChR allows for activation of NMDAR postsynaptically at synapses lacking AMPAR. Here we used dual immunolabeling and electron microscopy to examine the distribution of α7nAChR relative to AMPAR (GluR1, GluR2, and GluR3 subunits combined) at excitatory synapses in somatosensory cortex of adult and 1-week-old rats. α7nAChR occurred discretely over most of the thick postsynaptic densities in all cortical layers of both age groups. AMPAR immunoreactivity was also detectable at most synapses; its distribution was independent of that of α7nAChR. In both age groups, approximately one-quarter of asymmetrical synapses were α7nAChR positive and AMPAR negative. The variability of postsynaptic α7nAChR labeling density was greater at postnatal day (PD) 7 than in adulthood, and PD 7 neuropil contained a subset of small AMPA receptor-negative synapses with a high density of α7nAChR immunoreactivity. These observations support the idea that acetylcholine receptors can aid in activating glutamatergic synapses and work together with AMPA receptors to mediate postsynaptic excitation throughout life. PMID:12077196

  19. A β-hairpin structure in a 13-mer peptide that binds α-bungarotoxin with high affinity and neutralizes its toxicity

    PubMed Central

    Scherf, Tali; Kasher, Roni; Balass, Moshe; Fridkin, Mati; Fuchs, Sara; Katchalski-Katzir, Ephraim

    2001-01-01

    Snake-venom α-bungarotoxin is a member of the α-neurotoxin family that binds with very high affinity to the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. The structure of the complex between α-bungarotoxin and a 13-mer peptide (WRYYESSLEPYPD) that binds the toxin with high affinity, thus inhibiting its interactions with AChR with an IC50 of 2 nM, has been solved by 1H-NMR spectroscopy. The bound peptide folds into a β-hairpin structure created by two antiparallel β-strands, which combine with the already existing triple-stranded β-sheet of the toxin to form a five-stranded intermolecular, antiparallel β-sheet. Peptide residues Y3P, E5P, and L8P have the highest intermolecular contact area, indicating their importance in the binding of α-bungarotoxin; W1P, R2P, and Y4P also contribute significantly to the binding. A large number of characteristic hydrogen bonds and electrostatic and hydrophobic interactions are observed in the complex. The high-affinity peptide exhibits inhibitory potency that is better than any known peptide derived from AChR, and is equal to that of the whole α-subunit of AChR. The high degree of sequence similarity between the peptide and various types of AChRs implies that the binding mode found within the complex might possibly mimic the receptor binding to the toxin. The design of the high-affinity peptide was based on our previous findings: (i) the detection of a lead peptide (MRYYESSLKSYPD) that binds α-bungarotoxin, using a phage-display peptide library, (ii) the information about the three-dimensional structure of α-bungarotoxin/lead-peptide complex, and (iii) the amino acid sequence analysis of different AChRs. PMID:11381118

  20. [Histochemical findings of and fine structural changes in motor endplates in diseases with neuromuscular transmission abnormalities].

    PubMed

    Yoshimura, Toshiro; Motomura, Masakatsu; Tsujihata, Mitsuhiro

    2011-07-01

    We herein review the histochemical findings and fine structural changes of motor endplates associated with diseases causing neuromuscular transmission abnormalities. In anti-acetylcholine receptor (AChR) antibody-positive myasthenia gravis (MG), type 2 fiber atrophy is observed, and the motor endplates show a reduction in the nerve terminal area, simplification of the postsynaptic membrane, decreased number of acetylcholine receptors, and deposition of immune complexes. In anti-MuSK antibody-positive MG, the fine structure shows a decrease in the postsynaptic membrane length, but the secondary synaptic cleft is preserved. There is no decrease in the number of AChRs, and there are no deposits of immune complexes at the motor endplates. Patients with Lambert-Eaton myasthenic syndrome show type 2 fiber atrophy, their motor endplates show a decrease in both the mean postsynaptic area and postsynaptic membrane length in the brachial biceps muscle. Congenital myasthenic syndrome with episodic apnea is characterized only by small-sized synaptic vesicles; the postsynaptic area is preserved. In subjects with congenital myasthenic syndrome with acetylcholinesterase deficiency, quantitative electron microscopy reveals a significant decrease in the nerve terminal size and presynaptic membrane length; further, the Schwann cell processes extend into the primary synaptic cleft, and partially or completely occlude the presynaptic membrane. The postsynaptic folds are degenerated, and associated with pinocytotic vesicles and labyrinthine membranous networks. Patients with slow-channel congenital myasthenia syndrome show type 1 fiber predominance, and their junctional folds are typically degenerated with widened synaptic space and loss of AChRs. Patients with AChR deficiency syndrome caused by recessive mutations in AChR subunits also show type 1 fiber predominance, and while most junctional folds are normal, some are simplified and have smaller than normal endplates. Rapsin and Mu

  1. Pathogenesis of myasthenia gravis: update on disease types, models, and mechanisms.

    PubMed

    Phillips, William D; Vincent, Angela

    2016-01-01

    Myasthenia gravis is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. This can be generalised or localised to certain muscle groups, and involvement of the bulbar and respiratory muscles can be life threatening. The pathogenesis of myasthenia gravis depends upon the target and isotype of the autoantibodies. Most cases are caused by immunoglobulin (Ig)G1 and IgG3 antibodies to the acetylcholine receptor (AChR). They produce complement-mediated damage and increase the rate of AChR turnover, both mechanisms causing loss of AChR from the postsynaptic membrane. The thymus gland is involved in many patients, and there are experimental and genetic approaches to understand the failure of immune tolerance to the AChR. In a proportion of those patients without AChR antibodies, antibodies to muscle-specific kinase (MuSK), or related proteins such as agrin and low-density lipoprotein receptor-related protein 4 (LRP4), are present. MuSK antibodies are predominantly IgG4 and cause disassembly of the neuromuscular junction by disrupting the physiological function of MuSK in synapse maintenance and adaptation. Here we discuss how knowledge of neuromuscular junction structure and function has fed into understanding the mechanisms of AChR and MuSK antibodies. Myasthenia gravis remains a paradigm for autoantibody-mediated conditions and these observations show how much there is still to learn about synaptic function and pathological mechanisms. PMID:27408701

  2. Pathogenesis of myasthenia gravis: update on disease types, models, and mechanisms

    PubMed Central

    Phillips, William D.; Vincent, Angela

    2016-01-01

    Myasthenia gravis is an autoimmune disease of the neuromuscular junction (NMJ) caused by antibodies that attack components of the postsynaptic membrane, impair neuromuscular transmission, and lead to weakness and fatigue of skeletal muscle. This can be generalised or localised to certain muscle groups, and involvement of the bulbar and respiratory muscles can be life threatening. The pathogenesis of myasthenia gravis depends upon the target and isotype of the autoantibodies. Most cases are caused by immunoglobulin (Ig)G1 and IgG3 antibodies to the acetylcholine receptor (AChR). They produce complement-mediated damage and increase the rate of AChR turnover, both mechanisms causing loss of AChR from the postsynaptic membrane. The thymus gland is involved in many patients, and there are experimental and genetic approaches to understand the failure of immune tolerance to the AChR. In a proportion of those patients without AChR antibodies, antibodies to muscle-specific kinase (MuSK), or related proteins such as agrin and low-density lipoprotein receptor-related protein 4 (LRP4), are present. MuSK antibodies are predominantly IgG4 and cause disassembly of the neuromuscular junction by disrupting the physiological function of MuSK in synapse maintenance and adaptation. Here we discuss how knowledge of neuromuscular junction structure and function has fed into understanding the mechanisms of AChR and MuSK antibodies. Myasthenia gravis remains a paradigm for autoantibody-mediated conditions and these observations show how much there is still to learn about synaptic function and pathological mechanisms. PMID:27408701

  3. [Rhabdomyosarcoma lysis by T cells expressing a human autoantibody based chimeric receptor targeting the fetal acetylcholine receptors].

    PubMed

    Gattenlöhner, S

    2006-01-01

    Rhabdomyosarcomas (RMSs) are the most frequent malignant soft tissue tumors of childhood. Since even aggressive multimodality treatments including autologous stem cell rescue have failed to improve the < 20 % overall survival rate of children with metastatic RMS, novel treatment approaches are urgently needed. Looking for potential targets for immunotherapies, we identified the gamma subunit of the fetal acetylcholine receptor (fAChR) as a specific and overexpressed membrane antigen in RMS. Additionally we established a duplex RT-PCR with simultaneous amplification of alpha and gamma subunit message of the fAChR and the quantification of both transcripts resulting in alpha/gammaAChR ratio > 1 was 100% sensitive in alveolar and embryonal rhabdomyosarcoma. Since the fAChR was the first extracellular tumor marker that can distinguish rhabdomyosarcomas from nonrhabdomyomatous tumors and from normal muscle and therefore implies, that the fAChR may be a target for immunotherapeutic strategies, we synthesized a scFv antibody fragment directed against the fAChR and enigineered both a Pseudomonas exotoxin A based immunotoxin as well as a chimeric T cell receptor composed of the antigen-binding domain of the scFv fragment joined to the signaling domain of the T cell receptor zeta chain. The interaction of fAChzeta-transduced T cells with several RMS cell lines but not with fAChR-negative controls induced strong T cell activation, characterized by secretion of high amounts of interferon-gamma. Moreover after co-incubations with RMS cell lines fAChRzeta-transduced T cells as well fAChR specific immunotoxin induced specific receptor-concentration dependent tumor cell lysis. Therefore, fAChRzeta-transduced T cells and the fAChR specific immunotoxin respectively are promising new tools for the immunotherapy of rhabdomyosarcomas and may provide an effective complementary approach to eradicate residual or metastatic RMS cells in patients, since 1. RMS-direceted chemotherapies

  4. Nicotinic receptors in addiction pathways.

    PubMed

    Leslie, Frances M; Mojica, Celina Y; Reynaga, Daisy D

    2013-04-01

    Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that consist of pentameric combinations of α and β subunits. These receptors are widely distributed throughout the brain and are highly expressed in addiction circuitry. The role of nAChRs in regulating neuronal activity and motivated behavior is complex and varies both in and among brain regions. The rich diversity of central nAChRs has hampered the characterization of their structure and function with use of classic pharmacological techniques. However, recent molecular approaches using null mutant mice with specific regional lentiviral re-expression, in combination with neuroanatomical and electrophysiological techniques, have allowed the elucidation of the influence of different nAChR types on neuronal circuit activity and behavior. This review will address the influence of nAChRs on limbic dopamine circuitry and the medial habenula-interpeduncular nucleus complex, which are critical mediators of reinforced behavior. Characterization of the mechanisms underlying regulation of addiction pathways by endogenous cholinergic transmission and by nicotine may lead to the identification of new therapeutic targets for treating tobacco dependence and other addictions. PMID:23247824

  5. Sequence polymorphism in acetylcholinesterase transcripts and genotyping survey of BmAChE1 in laboratory and Mexican strains of Rhipicephalus (Boophilus) microplus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BmAChE1, BmAChE2, and BmAChE3 cDNAs of Rhipicephalus (Boophilus) microplus were sequenced and found to exhibit significant polymorphism. A portion of the predicted amino acid substitutions in BmAChE1, BmAChE2 and BmAChE3 were found predominantly in organophosphate-resistant (OP-R) strains, but most ...

  6. Genetics Home Reference: myasthenia gravis

    MedlinePlus

    ... AChR); in others, the antibodies attack a related protein called muscle-specific kinase (MuSK). In both cases, the abnormal antibodies lead to a reduction of available AChR. The AChR protein is critical for signaling between nerve and muscle ...

  7. The structure-AChE inhibitory activity relationships study in a series of pyridazine analogues.

    PubMed

    Saracoglu, M; Kandemirli, F

    2009-07-01

    The structure-activity relationships (SAR) are investigated by means of the Electronic-Topological Method (ETM) followed by the Neural Networks application (ETM-NN) for a class of anti-cholinesterase inhibitors (AChE, 53 molecules) being pyridazine derivatives. AChE activities of the series were measured in IC(50) units, and relative to the activity levels, the series was partitioned into classes of active and inactive compounds. Based on pharmacophores and antipharmacophores calculated by the ETM-software as sub-matrices containing important spatial and electronic characteristics, a system for the activity prognostication is developed. Input data for the ETM were taken as the results of conformational and quantum-mechanics calculations. To predict the activity, we used one of the most well known neural networks, namely, the feed-forward neural networks (FFNNs) trained with the back propagation algorithm. The supervised learning was performed using a variant of FFNN known as the Associative Neural Networks (ASNN). The result of the testing revealed that the high ETM's ability of predicting both activity and inactivity of potential AChE inhibitors. Analysis of HOMOs for the compounds containing Ph1 and APh1 has shown that atoms with the highest values of the atomic orbital coefficients are mainly those atoms that enter into the pharmacophores. Thus, the set of pharmacophores and antipharmacophores found as the result of this study forms a basis for a system of the anti-cholinesterase activity prediction. PMID:19689389

  8. Acetylcholinesterase Inhibitors (AChEI's) for the treatment of visual hallucinations in schizophrenia: a case report

    PubMed Central

    2010-01-01

    Background Visual hallucinations are commonly seen in various neurological and psychiatric disorders including schizophrenia. Current models of visual processing and studies in diseases including Parkinsons Disease and Lewy Body Dementia propose that Acetylcholine (Ach) plays a pivotal role in our ability to accurately interpret visual stimuli. Depletion of Ach is thought to be associated with visual hallucination generation. AchEI's have been used in the targeted treatment of visual hallucinations in dementia and Parkinson's Disease patients. In Schizophrenia, it is thought that a similar Ach depletion leads to visual hallucinations and may provide a target for drug treatment Case Presentation We present a case of a patient with Schizophrenia presenting with treatment resistant and significantly distressing visual hallucinations. After optimising treatment for schizophrenia we used Rivastigmine, an AchEI, as an adjunct to treat her symptoms successfully. Conclusions This case is the first to illustrate this novel use of an AchEI in the targeted treatment of visual hallucinations in a patient with Schizophrenia. Targeted therapy of this kind can be considered in challenging cases although more evidence is required in this field. PMID:20822516

  9. Impairment of contextual fear extinction by chronic nicotine and withdrawal from chronic nicotine is associated with hippocampal nAChR upregulation.

    PubMed

    Kutlu, Munir Gunes; Oliver, Chicora; Huang, Peng; Liu-Chen, Lee-Yuan; Gould, Thomas J

    2016-10-01

    Chronic nicotine and withdrawal from chronic nicotine have been shown to be major modulators of fear learning behavior. Moreover, recent studies from our laboratory have shown that acute nicotine impaired fear extinction and safety learning in mice. However, the effects of chronic nicotine and withdrawal on fear extinction are unknown. Therefore, the current experiments were conducted to investigate the effects of chronic nicotine as well as withdrawal from chronic nicotine on contextual fear extinction in mice. C57BL6/J mice were given contextual fear conditioning training and retention testing during chronic nicotine administration. Mice then received contextual fear extinction either during chronic nicotine or during withdrawal from chronic nicotine. Our results showed that contextual fear extinction was impaired both during chronic nicotine administration and subsequent withdrawal. However, it was also observed that the effects of prior chronic nicotine disappeared after 72 h in withdrawal, a timeline that closely matches with the timing of the chronic nicotine-induced upregulation of hippocampal nicotinic acetylcholine receptor (nAChR) density. Additional experiments found that 4 days, but not 1 day, of continuous nicotine administration upregulated hippocampal nAChRs and impaired contextual fear extinction. These effects disappeared following 72 h withdrawal. Overall, these experiments provide a potential link between nicotine-induced upregulation of hippocampal nAChRs and fear extinction deficits observed in patients with anxiety disorders, which may lead to advancements in the pharmacological treatment methods for this disorder. PMID:27378334

  10. APS8, a Polymeric Alkylpyridinium Salt Blocks α7 nAChR and Induces Apoptosis in Non-Small Cell Lung Carcinoma

    PubMed Central

    Zovko, Ana; Viktorsson, Kristina; Lewensohn, Rolf; Kološa, Katja; Filipič, Metka; Xing, Hong; Kem, William R.; Paleari, Laura; Turk, Tom

    2013-01-01

    Naturally occurring 3-alkylpyridinium polymers (poly-APS) from the marine sponge Reniera sarai, consisting of monomers containing polar pyridinium and nonpolar alkyl chain moieties, have been demonstrated to exert a wide range of biological activities, including a selective cytotoxicity against non-small cell lung cancer (NSCLC) cells. APS8, an analog of poly-APS with defined alkyl chain length and molecular size, non-competitively inhibits α7 nicotinic acetylcholine receptors (nAChRs) at nanomolar concentrations that are too low to be acetylcholinesterase (AChE) inhibitory or generally cytotoxic. In the present study we show that APS8 inhibits NSCLC tumor cell growth and activates apoptotic pathways. APS8 was not toxic for normal lung fibroblasts. Furthermore, in NSCLC cells, APS8 reduced the adverse anti-apoptotic, proliferative effects of nicotine. Our results suggest that APS8 or similar compounds might be considered as lead compounds to develop antitumor therapeutic agents for at least certain types of lung cancer. PMID:23880932

  11. Desensitizing and non-desensitizing subtypes of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in cockroach neurons.

    PubMed

    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

  12. Nitrosamines as nicotinic receptor ligands.

    PubMed

    Schuller, Hildegard M

    2007-05-30

    Nitrosamines are carcinogens formed in the mammalian organism from amine precursors contained in food, beverages, cosmetics and drugs. The potent carcinogen, NNK, and the weaker carcinogen, NNN, are nitrosamines formed from nicotine. Metabolites of the nitrosamines react with DNA to form adducts responsible for genotoxic effects. We have identified NNK as a high affinity agonist for the alpha7 nicotinic acetylcholine receptor (alpha7nAChR) whereas NNN bound with high affinity to epibatidine-sensitive nAChRs. Diethylnitrosamine (DEN) bound to both receptors but with lower affinity. High levels of the alpha7nAChR were expressed in human small cell lung cancer (SCLC) cell lines and in hamster pulmonary neuroendocrine cells (PNECs), which serve as a model for the cell of origin of human SCLC. Exposure of SCLC or PNECs to NNK or nicotine increased expression of the alpha7nAChR and caused influx of Ca(2+), activation of PKC, Raf-1, ERK1/2, and c-myc, resulting in the stimulation of cell proliferation. Signaling via the alpha7nAChR was enhanced when cells were maintained in an environment of 10-15% CO(2) similar to that in the diseased lung. Hamsters with hyperoxia-induced pulmonary fibrosis developed neuroendocrine lung carcinomas similar to human SCLC when treated with NNK, DEN, or nicotine. The development of the NNK-induced tumors was prevented by green tea or theophylline. The beta-adrenergic receptor agonist, isoproterenol or theophylline blocked NNK-induced cell proliferation in vitro. NNK and nicotine-induced hyperactivity of the alpha7nAChR/RAF/ERK1/2 pathway thus appears to play a crucial role in the development of SCLC in smokers and could be targeted for cancer prevention. PMID:17459420

  13. The value of acetylcholine receptor antibody in children with postural tachycardia syndrome.

    PubMed

    Li, Jiawei; Zhang, Qingyou; Liao, Ying; Zhang, Chunyu; Hao, Hongjun; Du, Junbao

    2015-01-01

    Postural tachycardia syndrome (POTS) is characterized by symptoms of orthostatic intolerance. Antibodies of acetylcholine receptor (AChR-ab) affect acetylcholine transmission between the ganglia and result in imbalance of the autonomic nervous system in POTS. This study was designed to analyze the clinical characteristics of POTS patients with AChR-ab positive and explore the value of AChR-ab in children with POTS. In 82 children with POTS, twenty patients (24.39%) were found as AChR-ab positive. Their clinical characteristics and hemodynamic responses to orthostatic changes were compared with the remaining 60 patients with negative AChR-ab. Symptoms of POTS children with AChR-ab positive were significantly severe than those of AChR-ab negative patients (p = 0.001). Preceding infection was predominant in patients with AChR-ab positive compared with that of patients with AChR-ab negative (p < 0.001). Syncope and fatigue were more common in the AChR-ab positive patients (p < 0.05). The change of upright heart rate was increased significantly in AChR-ab positive patients compared with AChR-ab negative cases (p = 0.013). Multiple logistic regression analysis revealed that preceding infection (OR 22.356, 95% CI 2.151-34.920), syncope (OR 11.570, 95% CI 2.098-63.810), and fatigue (OR 11.145, 95% CI 1.658-74.911) were independent risk factors for POTS with AChR-ab positive. In conclusion, POTS with positive AChR-ab was a heterogeneous disorder. Preceding infection, syncope and fatigue were their main clinical characteristics. PMID:25087056

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChR) are ligand-gated cation channels found throughout the body, and serve to mediate diverse physiological functions. Muscle-type nAChR located in the motor endplate region of muscle fibers play an integral role in muscle contraction and thus motor function. The...

  15. Allosteric modulation of M1 muscarinic acetylcholine receptor internalization and subcellular trafficking.

    PubMed

    Yeatman, Holly R; Lane, J Robert; Choy, Kwok Ho Christopher; Lambert, Nevin A; Sexton, Patrick M; Christopoulos, Arthur; Canals, Meritxell

    2014-05-30

    Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands. PMID:24753247

  16. Identification and Expression of Acetylcholinesterase in Octopus vulgaris Arm Development and Regeneration: a Conserved Role for ACHE?

    PubMed

    Fossati, Sara Maria; Candiani, Simona; Nödl, Marie-Therese; Maragliano, Luca; Pennuto, Maria; Domingues, Pedro; Benfenati, Fabio; Pestarino, Mario; Zullo, Letizia

    2015-08-01

    Acetylcholinesterase (ACHE) is a glycoprotein with a key role in terminating synaptic transmission in cholinergic neurons of both vertebrates and invertebrates. ACHE is also involved in the regulation of cell growth and morphogenesis during embryogenesis and regeneration acting through its non-cholinergic sites. The mollusk Octopus vulgaris provides a powerful model for investigating the mechanisms underlying tissue morphogenesis due to its high regenerative power. Here, we performed a comparative investigation of arm morphogenesis during adult arm regeneration and embryonic arm development which may provide insights on the conserved ACHE pathways. In this study, we cloned and characterized O. vulgaris ACHE, finding a single highly conserved ACHE hydrophobic variant, characterized by prototypical catalytic sites and a putative consensus region for a glycosylphosphatidylinositol (GPI)-anchor attachment at the COOH-terminus. We then show that its expression level is correlated to the stage of morphogenesis in both adult and embryonic arm. In particular, ACHE is localized in typical neuronal sites when adult-like arm morphology is established and in differentiating cell locations during the early stages of arm morphogenesis. This possibility is also supported by the presence in the ACHE sequence and model structure of both cholinergic and non-cholinergic sites. This study provides insights into ACHE conserved roles during processes of arm morphogenesis. In addition, our modeling study offers a solid basis for predicting the interaction of the ACHE domains with pharmacological blockers for in vivo investigations. We therefore suggest ACHE as a target for the regulation of tissue morphogenesis. PMID:25112677

  17. nAChRs Mediate Human Embryonic Stem Cell-Derived Endothelial Cells: Proliferation, Apoptosis, and Angiogenesis

    PubMed Central

    Velotta, Jeffrey B.; Huang, Mei; Li, Zongjin; Lee, Andrew; Robbins, Robert C.; Cooke, John P.; Wu, Joseph C.

    2009-01-01

    Background Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs). Methods and Results To induce endothelial cell differentiation, undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days, CD31+ cells (13.7±2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation, these hESC-ECs expressed endothelial specific markers such as vWF (96.3±1.4%), CD31 (97.2±2.5%), and VE-cadherin (93.7±2.8%), form vascular-like channels, and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward, 5×106 hESC-ECs treated for 24 hours with nicotine (10−8 M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 µg/ml) in the drinking water. Surprisingly, bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally, in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs). Conclusions This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs, and enhance their angiogenic effects in vivo. Furthermore, activation of nAChRs has anti-apoptotic, angiogenic, and proliferative effects through MAPK and Akt signaling pathways. PMID:19753305

  18. Allosteric Modulation of Muscarinic Acetylcholine Receptors

    PubMed Central

    Gregory, Karen J; Sexton, Patrick M; Christopoulos, Arthur

    2007-01-01

    Muscarinic acetylcholine receptors (mAChRs) are prototypical Family A G protein coupled-receptors. The five mAChR subtypes are widespread throughout the periphery and the central nervous system and, accordingly, are widely involved in a variety of both physiological and pathophysiological processes. There currently remains an unmet need for better therapeutic agents that can selectively target a given mAChR subtype to the relative exclusion of others. The main reason for the lack of such selective mAChR ligands is the high sequence homology within the acetylcholine-binding site (orthosteric site) across all mAChRs. However, the mAChRs possess at least one, and likely two, extracellular allosteric binding sites that can recognize small molecule allosteric modulators to regulate the binding and function of orthosteric ligands. Extensive studies of prototypical mAChR modulators, such as gallamine and alcuronium, have provided strong pharmacological evidence, and associated structure-activity relationships (SAR), for a “common” allosteric site on all five mAChRs. These studies are also supported by mutagenesis experiments implicating the second extracellular loop and the interface between the third extracellular loop and the top of transmembrane domain 7 as contributing to the common allosteric site. Other studies are also delineating the pharmacology of a second allosteric site, recognized by compounds such as staurosporine. In addition, allosteric agonists, such as McN-A-343, AC-42 and N-desmethylclozapine, have also been identified. Current challenges to the field include the ability to effectively detect and validate allosteric mechanisms, and to quantify allosteric effects on binding affinity and signaling efficacy to inform allosteric modulator SAR. PMID:19305798

  19. Inhibition of the acetylcholine receptor by histrionicotoxin.

    PubMed Central

    Anwyl, R.; Narahashi, T.

    1980-01-01

    1 The action of C5-decahydrohistrionicotoxin (C5-HTX) has been investigated on the extrajunctional acetylcholine (ACh) receptors of denervated rat muscle. 2 C5-HTX causes both a rapid and slow reduction in amplitude of iontophoretic ACh potentials evoked at all frequencies from the extrajunctional receptors. 3 C5-HTX also causes a time-dependent inhibition of the iontophoretic potentials evoked at frequencies greater than 0.02 Hz. This inhibition was observed either alone or superimposed upon desensitization, and may be caused by a similar mechanism to desensitization. PMID:7378635

  20. Aporphine metho salts as neuronal nicotinic acetylcholine receptor blockers.

    PubMed

    Iturriaga-Vásquez, Patricio; Pérez, Edwin G; Slater, E Yvonne; Bermúdez, Isabel; Cassels, Bruce K

    2007-05-15

    (S)-Aporphine metho salts with the 1,2,9,10 oxygenation pattern displaced radioligands from recombinant human alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors (nAChR) at low micromolar concentrations. The affinity of the nonphenolic glaucine methiodide (4) (vs [(3)H]cytisine) was the lowest at alpha4beta2 nAChR (K(i)=10 microM), and predicentrine methiodide (2) and xanthoplanine iodide (3), with free hydroxyl groups at C-2 or C-9, respectively, had the highest affinity at these receptors (K(i) approximately 1 microM), while the affinity of the diphenolic boldine methiodide (1) was intermediate between these values. At homomeric alpha7 nAChR, xanthoplanine had the highest affinity (K(i)=10 microM) vs [(125)I]alpha-bungarotoxin while the other three compounds displaced the radioligand with K(i) values between 15 and 21 microM. At 100 microM, all four compounds inhibited the responses of these receptors to EC(50) concentrations of ACh. The effects of xanthoplanine iodide (3) were studied in more detail. Xanthoplanine fully inhibited the EC(50) ACh responses of both alpha7 and alpha4beta2 nACh receptors with estimated IC(50) values of 9+/-3 microM (alpha7) and 5+/-0.8 microM (alpha4beta2). PMID:17391965

  1. Differential effects of lysophosphatidylcholine and ACh on muscarinic K(+),non-selective cation and Ca(2+) currents in guinea-pig atrial cells.

    PubMed

    Li, Libing; Matsuoka, Isao; Sakamoto, Kazuho; Kimura, Junko

    2016-06-01

    We compared the effects of lysophosphatidylcholine (LPC) and acetylcholine (ACh) on IK(ACh), ICa and a non-selective cation current (INSC) in guinea-pig atrial myocytes to clarify whether LPC and ACh activate similar Gi/o-coupled effector systems. IK(ACh), ICa and INSC were analyzed in single atrial myocytes by the whole cell patch-clamp. LPC induced INSC in a concentration-dependent manner in atrial cells. ACh activated IK(ACh), but failed to evoke INSC. LPC also activated IK(ACh) but with significantly less potency than ACh. The effects of both ligands on IK(ACh) were inhibited by intracellular loading of pre-activated PTX. This treatment also inhibited LPC-induced INSC, indicating that IK(ACh) and INSC induced by LPC are both mediated by Gi/o. LPC and ACh had similar potencies in inhibiting ICa, which was pre-augmented by forskolin, indicating that LPC and ACh activate similar amounts of α-subunits of Gi/o. The different effects of LPC and ACh on IK(ACh) and INSC may suggest that LPC and ACh activate Gi/o having different types of βγ subunits, and that LPC-induced INSC may be mediated by βγ subunits of Gi/o, which are less effective in inducing IK(ACh). PMID:26911304

  2. Nerve fibers that were not stained with the non-specific acetylcholinesterase (NsAchE) method, and TRPV1- and IB4-positive nerve fibers in the rat cornea.

    PubMed

    Nakagawa, Hiroshi; Hiura, Akio; Mitome, Masato; Ishimura, Kazunori

    2009-08-01

    Previously, we noticed the presence of nerve fiber-like structures in a whole mount preparation of the rat cornea that had not been stained with the non-specific acetylcholinesterase (NsAchE) method. These nerve-like fibers were projected into the central area of the cornea, forming a mesh-like pattern. The aim of this study is to examine the properties of these mesh-like fibers using the following two methods: their sensitivity to capsaicin and the detection of isolectin B4 (IB4)- and capsaicin receptor TRPV1 (transient receptor potential vanilloid 1)-reactivities. The mean disappeared area of non-stained fibers after NsAchE treatment was 26% of the total areas in the neonatally capsaicin-treated cornea. Bunches composed of fine IB4-positive nerve fibers were seen in a whole mount preparation. There were connections between the bunches, producing a mesh-like pattern similar to that of the fibers that were not stained with NsAchE. Fine TRPV1-immunoreactive (ir) nerve fibers were also shown to form bunches, with connections between each bunch observed in whole mount preparations. Thus, TRPV1-ir nerve fibers seem to densely innervate the rat corneal subepithelial stroma and are distinct from the NsAchE-positive nerve fibers. The TRPV1-ir fine nerve fibers overlapped with the IB4-positive nerve fibers, suggesting that the mesh-like fibers that were not stained with NsAchE are fine nociceptive sensory nerve fibers because of their sensitivity to capsaicin and similar distribution pattern to IB4- and TRPV1-positive nerve fibers. PMID:19763029

  3. Erosion of a-C:H films under interaction with nitrous oxide afterglow discharge

    NASA Astrophysics Data System (ADS)

    Zalavutdinov, R. Kh.; Gorodetsky, A. E.; Bukhovets, V. L.; Zakharov, A. P.; Mazul, I. V.

    2009-06-01

    Hydrocarbon film removal using chemically active oxygen formed in a direct current glow discharge with a hollow cathode in nitrous oxide was investigated. In the afterglow region sufficiently fast removal of a-C:H films about 500 nm thick during about 8 h was achieved at N 2O pressure of 12 Pa and 370 K. The erosion rate in the afterglow region was directly proportional to the initial pressure and increased two orders of magnitude at temperature rising from 300 to 500 K. The products of a-C:H film plasmolysis were CO, CO 2, H 2O, and H 2. After removal of a-C:H films previously deposited on stainless steel, molybdenum or tungsten 3-30 nm thick oxide films were formed on the substrates. Reactions of oxygen ion neutralization and atomic oxygen recombination suppressed further oxidation of the materials.

  4. α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit

    PubMed Central

    Mackey, Elisha D.W.; Engle, Staci E.; Kim, Mi Ran; O’Neill, Heidi C.; Wageman, Charles R.; Patzlaff, Natalie E.; Wang, Ying; Grady, Sharon R.; McIntosh, J. Michael; Marks, Michael J.; Lester, Henry A.; Drenan, Ryan M.

    2012-01-01

    Nicotinic ACh receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus coeruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with GFP fused in-frame in the M3–M4 intracellular loop. Inα6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitiveα6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway. PMID:22836257

  5. Role of α5 Nicotinic Acetylcholine Receptors in Pharmacological and Behavioral Effects of Nicotine in Mice

    PubMed Central

    Marks, M. J.; Vann, R. E.; Chen, X.; Gamage, T. F.; Warner, J. A.; Damaj, M. I.

    2010-01-01

    Incorporation of the α5 nicotinic acetylcholine receptor (nAChR) subunit can greatly influence nAChR function without altering receptor number. Although few animal studies have assessed the role of the α5 nAChR in nicotine-mediated behaviors, recent evidence suggests an association between polymorphisms in the α5 nAChR gene and nicotine dependence phenotypes in humans. Thus, additional studies are imperative to elucidate the role and function of the α5 nAChR subunit in nicotine dependence. Using α5(−/−) mice, the current study aimed to examine the role of α5 nAChRs in the initial pharmacological effects of nicotine, nicotine reward using the conditioned place preference model, and the discriminative effects of nicotine using a two-lever drug discrimination model. 86Rb+ efflux and 125I-epibatidine binding assays were conducted to examine the effect of α5 nAChR subunit deletion on expression and activity of functional nAChRs. Results show that α5(−/−) mice are less sensitive to the initial effects of nicotine in antinociception, locomotor activity, and hypothermia measures and that the α5 nAChR is involved in nicotine reward. Alternatively, α5(−/−) mice did not differ from wild-type littermates in sensitivity to the discriminative stimulus effects of nicotine. Furthermore, deletion of the α5 nAChR subunit resulted in a statistically significant decrease in function in the thalamus and hindbrain, but the decreases noted in spinal cord were not statistically significant. Receptor number was unaltered in all areas tested. Taken together, results of the study suggest that α5 nAChRs are involved in nicotine-mediated behaviors relevant to development of nicotine dependence. PMID:20400469

  6. A novel 4/6-type alpha-conotoxin ViIA selectively inhibits nAchR α3β2 subtype.

    PubMed

    Li, Liang; Liu, Na; Ding, Rong; Wang, Shuo; Liu, Zhuguo; Li, Haiying; Zheng, Xing; Dai, Qiuyun

    2015-12-01

    Conotoxins (CTxs) are typically small peptides composed of 12-50 amino acid residues with 2-5 disulfide bridges. Most of them potently and selectively target a wide variety of ion channels and membrane receptors. They are highly valued as neuropharmacological probes and in pharmaceutical development. In this work, a novel α4/6-CTx named ViIA (RDCCSNPPCAHNNPDC-NH2) was identified from a cDNA library of the venom ducts of Conus virgo (C. virgo). ViIA was then synthesized chemically and its disulfide connectivity was identified as 'C(1)-C(3), C(2)-C(4)'. Its molecular targets were further assessed using two-electrode voltage clamping. The results indicated that ViIA selectively inhibited nicotinic acetylcholine receptor (nAChR) α3β2 subtype with an IC50 of 845.5 nM, but did not target dorsal root ganglion sodium (Na(+))-, potassium (K(+))- or calcium (Ca(2+))-ion channels. Further structure-activity relationship analysis demonstrated that Arg(1) and His(11) but not Asp(2) were the functional residues. To the best of our knowledge, ViIA is the first 4/6 α-CTx that selectively inhibits nAChR α3β2 subtype. This finding expands the knowledge of targets of α4/6-family CTxs. PMID:26511093

  7. Therapeutic Potential of α7 Nicotinic Acetylcholine Receptors.

    PubMed

    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

  8. A new family of insect muscarinic acetylcholine receptors.

    PubMed

    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

  9. Functional analysis of Torpedo californica nicotinic acetylcholine receptors in multiple activation states by SSM-based electrophysiology.

    PubMed

    Niessen, K V; Muschik, S; Langguth, F; Rappenglück, S; Seeger, T; Thiermann, H; Worek, F

    2016-04-15

    Organophosphorus compounds (OPC), i.e. nerve agents or pesticides, are highly toxic due to their strong inhibition potency against acetylcholinesterase (AChE). Inhibited AChE results in accumulation of acetylcholine in the synaptic cleft and thus the desensitisation of the nicotinic acetylcholine receptor (nAChR) in the postsynaptic membrane is provoked. Direct targeting of nAChR to reduce receptor desensitisation might be an alternative therapeutic approach. For drug discovery, functional properties of potent therapeutic candidates need to be investigated in addition to affinity properties. Solid supported membrane (SSM)-based electrophysiology is useful for functional characterisation of ligand-gated ion channels like nAChRs, as charge translocations via capacitive coupling of the supporting membrane can be measured. By varying the agonist (carbamoylcholine) concentration, different functional states of the nAChR were initiated. Using plasma membrane preparations obtained from Torpedo californica electric organ, functional properties of selected nAChR ligands and non-oxime bispyridinium compounds were investigated. Depending on overall-size, the bispyridinium compounds enhanced or inhibited cholinergic signals induced by 100μM carbamoylcholine. Applying excessive concentrations of the agonist carbamoylcholine provoked desensitisation of the nAChRs, whereas addition of bispyridinium compounds bearing short alkyl linkers exhibited functional recovery of previously desensitised nAChRs. The results suggest that these non-oxime bispyridinium compounds possibly interacted with nAChR subtypes in a manner of a positive allosteric modulator (PAM). The described newly developed functional assay is a valuable tool for the assessment of functional properties of potential compounds such as nAChR modulating ligands, which might be a promising approach in the therapeutically treatment of OPC-poisonings. PMID:26851639

  10. Nicotinic acetylcholine receptor-mediated responses in medial vestibular and prepositus hypoglossi nuclei neurons showing distinct neurotransmitter phenotypes.

    PubMed

    Zhang, Yue; Yanagawa, Yuchio; Saito, Yasuhiko

    2016-06-01

    Cholinergic transmission in both the medial vestibular nucleus (MVN) and prepositus hypoglossi nucleus (PHN) plays an important role in horizontal eye movements. We previously demonstrated that the current responses mediated via nicotinic acetylcholine receptors (nAChRs) were larger than those mediated via muscarinic acetylcholine receptors (mAChRs) in cholinergic MVN and PHN neurons that project to the cerebellum. In this study, to clarify the predominant nAChR responses and the expression patterns of nAChRs in MVN and PHN neurons that exhibit distinct neurotransmitter phenotypes, we identified cholinergic, inhibitory, and glutamatergic neurons using specific transgenic rats and investigated current responses to the application of acetylcholine (ACh) using whole cell recordings in brain stem slices. ACh application induced larger nAChR-mediated currents than mAChR-mediated currents in every neuronal phenotype. In the presence of an mAChR antagonist, we found three types of nAChR-mediated currents that exhibited different rise and decay times and designated these as fast (F)-, slow (S)-, and fast and slow (FS)-type currents. F-type currents were the predominant response in inhibitory MVN neurons, whereas S-type currents were observed in the majority of glutamatergic MVN and PHN neurons. No dominant response type was observed in cholinergic neurons. Pharmacological analyses revealed that the F-, S-, and FS-type currents were mainly mediated by α7, non-α7, and both α7 and non-α7 nAChRs, respectively. These findings suggest that cholinergic responses in the major neuronal populations of the MVN and PHN are predominantly mediated by nAChRs and that the expression of α7 and non-α7 nAChRs differ among the neuronal phenotypes. PMID:26936981

  11. Myasthenia gravis, a model of organ-specific autoimmune disease.

    PubMed

    Berrih-Aknin, S

    1995-04-01

    Myasthenia gravis (MG) is a neuromuscular disorder of autoimmune origin. Most patients have antibodies directed against the acetylcholine receptor (AChR) that interfere with neuromuscular transmission. MG is a model of organ-specific autoimmune disease in which the autoantigen, AChR, is well characterized. However, several questions remain unanswered. Why is AChR, which is present in the thymus, not tolerized? Why does the anti-AChR antibody titre not correlate with clinical manifestations, and why do some patients not have such antibodies? What genetic elements are involved in disease susceptibility? How is the expression of AChR regulated after its attack by autoantibodies? Could MG patients benefit from new immunomodulatory treatments? At the IVth EuroMyasthenia meeting, held in Versailles in April 1994, almost 200 researchers and physicians met to discuss the pathophysiology and treatment of myasthenia gravis and to provide answers to some of these questions. The sessions covered immunological, genetic and clinical aspects of MG. PMID:7612144

  12. The Conserved RIC-3 Coiled-Coil Domain Mediates Receptor-specific Interactions with Nicotinic Acetylcholine Receptors

    PubMed Central

    Biala, Yoav; Liewald, Jana F.; Ben-Ami, Hagit Cohen; Gottschalk, Alexander

    2009-01-01

    RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function. To examine the role of CC-I, we studied effects of its deletion on Caenorhabditis elegans nAChRs in vivo. Presence of CC-I promoted maturation of particular nAChRs expressed in body-wall muscle, whereas it was not required for other nAChR subtypes expressed in neurons or pharyngeal muscles. This effect is receptor-specific, because it could be reproduced after heterologous expression. Consistently, coimmunoprecipitation analysis showed that CC-I enhances the interaction of RIC-3 with a nAChR that requires CC-I in vivo; thus CC-I appears to enhance affinity of RIC-3 to specific nAChRs. However, we found that this function of CC-I is redundant with functions of sequences downstream to CC-I, potentially a second coiled-coil. Alternative splicing in both vertebrates and invertebrates generates RIC-3 transcripts that lack the entire C-terminus, or only CC-I. Thus, our results suggest that RIC-3 alternative splicing enables subtype specific regulation of nAChR maturation. PMID:19116311

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

    PubMed Central

    Kudryavtsev, Denis; Shelukhina, Irina; Vulfius, Catherine; Makarieva, Tatyana; Stonik, Valentin; Zhmak, Maxim; Ivanov, Igor; Kasheverov, Igor; Utkin, Yuri; Tsetlin, Victor

    2015-01-01

    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. PMID:26008231

  14. Down-regulation of muscarinic receptors and the m3 subtype in white-footed mice by dietary exposure to parathion

    USGS Publications Warehouse

    Jett, D.A.; Hill, E.F.; Fernando, J.C.; Eldefrawi, M.E.; Eldefrawi, A.T.

    1993-01-01

    The effect of ad libitum dietary exposure (as occurs in the field) to parathion for 14 d was investigated on the muscarinic acetylcholine receptor (mAChR) in brains and submaxillary glands of adults of a field species, the white-footed mouse Peromyscus leucopus. Immunoprecipitation using subtype selective antibodies revealed that the relative ratios of the m1-m5 mAChR subtypes in Peromyscus brain were similar to those in rat brain. There was little variability in acetylcholinesterase (AChE) activity in control mice brains but large variability in 39 exposed mice, resulting from differences in food ingestion and parathion metabolism. Accordingly, data on radioligand binding to mAChRs in each mouse brain were correlated with brain AChE activity in the same mouse, and AChE inhibition served as a biomarker of exposure reflecting in situ paraoxon concentrations. Exposure to parathion for 14 d reduced maximal binding (Bmax) of [3H]quinuclidinyl benzilate ([3H]QNB), [3H]-N-methylscopolamine ([3H]NMS), and [3H]-4-diphenylacetoxy-N-methylpiperidine methiodide ([3H]-4-DAMP) by up to approximately 58% without affecting receptor affinities for these ligands. Maximal reduction in Bmax of [3H]QNB and [3H]-4-DAMP binding occurred in mice with highest AChE inhibition, while equivalent maximal reduction in Bmax of [3H]NMS occurred in mice with only approximately 10% AChE inhibition, without further change at higher parathion doses. This is believed to be due to the hydrophilicity of [3H]NMS, which limits its accessibility to internalized desensitized receptors. In submaxillary glands (mAChRs are predominantly m3 subtype), there were significant dose-dependent reductions in [3H]QNB binding and m3 mRNA levels in exposed mice, revealed by Northern blot analyses. The reduction in m3 receptors is suggested to result mostly from reduced synthesis at the transcription level, rather than from translational or posttranslational events. The data suggest that down-regulation of mAChRs occurs

  15. The role of the a7 subunit of the nicotinic acetylcholine receptor in the acute toxicosis of methyllycaconitine in mice.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The adverse physiological effects of methyllycaconitine (MLA) have been attributed to its competitive antagonism of nicotinic acetylcholine receptors (nAChRs). Recent research demonstrated a correlation between the LD50 of MLA and the amount of a7 nAChR in various mouse strains, suggesting that mice...

  16. Putative nicotinic acetylchloline receptor subunits express differentially through life cycle of codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nicotinic acetylcholine receptors (nAChRs) are the targets of neonicotinoids and spinosads, two insecticides used in orchards to effectively control codling moth, Cydia pomonella (L.)(Lepidoptera: Tortricidae). The nAChRs mediate the fast actions of the neurotransmitter acetylcholine in synaptic tr...

  17. α6β2*-subtype nicotinic acetylcholine receptors are more sensitive than α4β2*-subtype receptors to regulation by chronic nicotine administration

    PubMed Central

    Marks, MJ; Grady, SR; Salminen, O; Paley, MA; Wageman, CR; McIntosh, JM; Whiteaker, P

    2014-01-01

    Nicotinic acetylcholine receptors (nAChR) of the α6β2* subtype (where * indicates the possible presence of additional subunits) are prominently expressed on dopaminergic neurons. Because of this, their role in tobacco use and nicotine dependence has received much attention. Previous studies have demonstrated that α6β2*-nAChR are downregulated following chronic nicotine exposure (unlike other subtypes that have been investigated – most prominently α4β2* nAChR). This study examines, for the first time, effects across a comprehensive chronic nicotine dose range. Chronic nicotine dose-responses and quantitative ligand-binding autoradiography were used to define nicotine sensitivity of changes in α4β2*-nAChR and α6β2*-nAChR expression. α6β2*-nAChR downregulation by chronic nicotine exposure in dopaminergic and optic-tract nuclei was ≈three-fold more sensitive than upregulation of α4β2*-nAChR. In contrast, nAChR-mediated [3H]-dopamine release from dopamine-terminal region synaptosomal preparations changed only in response to chronic treatment with high nicotine doses, while dopaminergic parameters (transporter expression and activity, dopamine receptor expression) were largely unchanged. Functional measures in olfactory tubercle preparations were made for the first time; both nAChR expression levels and nAChR-mediated functional measures changed differently between striatum and olfactory tubercles. These results show that functional changes measured using synaptosomal [3H]-DA release are primarily due to changes in nAChR, rather than in dopaminergic, function. PMID:24661093

  18. Muscarinic acetylcholine receptor modulation of mu (mu) opioid receptors in adult rat sphenopalatine ganglion neurons.

    PubMed

    Margas, Wojciech; Mahmoud, Saifeldin; Ruiz-Velasco, Victor

    2010-01-01

    The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow. PMID:19889856

  19. Role of the nicotinic acetylcholine receptor in Alzheimer's disease pathology and treatment.

    PubMed

    Lombardo, Sylvia; Maskos, Uwe

    2015-09-01

    Alzheimer's Disease (AD) is the major form of senile dementia, characterized by neuronal loss, extracellular deposits, and neurofibrillary tangles. It is accompanied by a loss of cholinergic tone, and acetylcholine (ACh) levels in the brain, which were hypothesized to be responsible for the cognitive decline observed in AD. Current medication is restricted to enhancing cholinergic signalling for symptomatic treatment of AD patients. The nicotinic acetylcholine receptor family (nAChR) and the muscarinic acetylcholine receptor family (mAChR) are the target of ACh in the brain. Both families of receptors are affected in AD. It was demonstrated that amyloid beta (Aβ) interacts with nAChRs. Here we discuss how Aβ activates or inhibits nAChRs, and how this interaction contributes to AD pathology. We will discuss the potential role of nAChRs as therapeutic targets. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'. PMID:25514383

  20. Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

    PubMed Central

    Schmidt, Nadine; Gajendran, Nadesan; Martinez-Pena y Valenzuela, Isabel; Wakefield, Sarah; Thurnheer, Raphael

    2011-01-01

    Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1. PMID:22184199

  1. Draft Genome Sequence of Aldehyde-Degrading Strain Halomonas axialensis ACH-L-8.

    PubMed

    Ye, Jun; Ren, Chong; Shan, Xiexie; Zeng, Runying

    2016-01-01

    Halomonas axialensisACH-L-8, a deep-sea strain isolated from the South China Sea, has the ability to degrade aldehydes. Here, we present an annotated draft genome sequence of this species, which could provide fundamental molecular information on the aldehydes-degrading mechanism. PMID:27081145

  2. Helium permeation through a-C:H films deposited on polymeric substrates

    NASA Astrophysics Data System (ADS)

    Valentini, L.; Bellachioma, M. C.; Lozzi, L.; Santucci, S.; Kenny, J. M.

    2002-09-01

    The influence of amorphous hydrogenated carbon a-C:H coatings on gas permeation through polymer films was investigated. Hydrogenated amorphous carbon (a-C:H) films were deposited, at room temperature, from a CH4/Ar plasma produced by a radio frequency glow discharge system at 13.56 MHz. Polyether-etherketone (PEEK) and polyetherimide foils with different thicknesses were used as substrates. The permeation of He was measured and the reduction of the permeability coefficient is correlated here to the composition and density of the a-C:H films. The density and film structure of the layers were analyzed using x-ray reflectivity and Raman spectroscopy of films deposited onto silicon reference samples. A less pronounced reduction of the permeability coefficients for hard, dense diamond-like layers is reported with respect to those obtained for soft, polymer-like layers on PEEK substrates. Surprisingly, the barrier efficacy of the coating decreases with an increase in a-C:H film density. This unexpected result is attributed to intrinsic stress and the corresponding formation of microcracks. The effect of nitrogen incorporation, which reduces film permeability, is investigated in terms of the stress relaxation mechanism promoted. copyright 2002 American Vacuum Society.

  3. Genome Sequence of the Mycorrhiza Helper Bacterium Streptomyces sp. Strain AcH 505

    PubMed Central

    Feldhahn, L.; Buscot, F.; Wubet, T.

    2015-01-01

    A draft genome sequence of Streptomyces sp. strain AcH 505 is presented here. The genome encodes 22 secondary metabolite gene clusters and a large arsenal of secreted proteins, and their comparative and functional analyses will help to advance our knowledge of symbiotic interactions and fungal and plant biomass degradation. PMID:25838498

  4. Draft Genome Sequence of Aldehyde-Degrading Strain Halomonas axialensis ACH-L-8

    PubMed Central

    Ye, Jun; Ren, Chong; Shan, Xiexie

    2016-01-01

    Halomonas axialensis ACH-L-8, a deep-sea strain isolated from the South China Sea, has the ability to degrade aldehydes. Here, we present an annotated draft genome sequence of this species, which could provide fundamental molecular information on the aldehydes-degrading mechanism. PMID:27081145

  5. Anti-MuSK autoantibodies block binding of collagen Q to MuSK

    PubMed Central

    Kawakami, Y.; Ito, M.; Hirayama, M.; Sahashi, K.; Ohkawara, B.; Masuda, A.; Nishida, H.; Mabuchi, N.; Engel, A.G.

    2011-01-01

    Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%–15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK–immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK. Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq−/− mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice. Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the size and density of AChR and MuSK. Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq−/− mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox. PMID:22013178

  6. The combination of memantine and galantamine improves cognition in rats: The synergistic role of the α7 nicotinic acetylcholine and NMDA receptors.

    PubMed

    Nikiforuk, Agnieszka; Potasiewicz, Agnieszka; Kos, Tomasz; Popik, Piotr

    2016-10-15

    The combination of memantine and acetylcholinesterase inhibitors (AChEIs) is used as a therapeutic strategy to improve cognition in Alzheimer's disease. Among AChEIs, galantamine, which is also a positive allosteric modulator (PAM) of nicotinic acetylcholine receptors (nAChRs), including α7-nAChRs, may be particularly beneficial. The α7-nAChR is involved in interactions between the cholinergic and glutamatergic systems. In the present study, we investigated the potential role of α7-nAChRs in the pro-cognitive effects of this drug combination. To this aim, cognitive performance in rats was assessed using the attentional set shifting task (ASST) and novel object recognition task (NORT). Co-administration of inactive doses of memantine with galantamine facilitated the rats' set-shifting performance and reversed delay-induced deficits in object recognition. These effects were blocked by the α7-nAChR antagonist methyllycaconitine, suggesting that the observed cognitive enhancement is α7-nAChR dependent. Moreover, combined administration of memantine with inactive doses of selective α7-nAChRs PAMs, CCMI and PNU-120596, also improved ASST and NORT performance in a methyllycaconitine-dependent manner. Stimulation of α7-nAChRs may underlie the pro-cognitive effects of combining memantine and galantamine. Our results suggest that memantine, when given with enhancers of α7-nAChRs, may represent an effective strategy for cognitive improvement. PMID:27435422

  7. α7 Nicotinic Receptor Agonists: Potential Therapeutic Drugs for Treatment of Cognitive Impairments in Schizophrenia and Alzheimer’s Disease

    PubMed Central

    Toyohara, Jun; Hashimoto, Kenji

    2010-01-01

    Accumulating evidence suggests that α7 nicotinic receptors (α7 nAChRs), a subtype of nAChRs, play a role in the pathophysiology of neuropsychiatric diseases, including schizophrenia and Alzheimer’s disease (AD). A number of psychopharmacological and genetic studies shown that α7 nAChRs play an important role in the deficits of P50 auditory evoked potential in patients with schizophrenia, and that (α nAChR agonists would be potential therapeutic drugs for cognitive impairments associated with P50 deficits in schizophrenia. Furthermore, some studies have demonstrated that α7 nAChRs might play a key role in the amyloid-β (Aβ)-mediated pathology of AD, and that α7 nAChR agonists would be potential therapeutic drugs for Aβ deposition in the brains of patients with AD. Interestingly, the altered expression of α7 nAChRs in the postmortem brain tissues from patients with schizophrenia and AD has been reported. Based on all these findings, selective α7 nAChR agonists can be considered potential therapeutic drugs for cognitive impairments in both schizophrenia and AD. In this article, we review the recent research into the role of α7 nAChRs in the pathophysiology of these diseases and into the potential use of novel α7 nAChR agonists as therapeutic drugs. PMID:21249164

  8. Functional interactions between the SK2 channel and the nicotinic acetylcholine receptor in enteric neurons of the guinea pig ileum.

    PubMed

    Nakajima, Hidemitsu; Goto, Hiroto; Azuma, Yasu-Taka; Fujita, Akikazu; Takeuchi, Tadayoshi

    2007-12-01

    The neurotransmitter acetylcholine (ACh) plays a critical role in gastrointestinal function. The role of the small conductance Ca2+-activated K+ (SK) channel in ACh release was examined using myenteric plexus preparations of guinea pig ileum. Apamin, an inhibitor of the SK channel, significantly enhanced nicotine-induced ACh release, but neither electrical field stimulation- nor 5-hydroxytryptamine-induced ACh release, suggesting that SK channels might be selectively involved in the regulation of nicotine-induced ACh release. Therefore, we investigated the distribution of SK2 and SK3 subunits and the interaction between SK2 channels and nicotinic ACh receptors (nAChRs) in the guinea pig ileum. The immunoreactivity of SK2 subunits was located in enteric neuronal cells. Furthermore, SK2-immunoreactive cells stained with an antibody for choline acetyltransferase, a marker for cholinergic neurons, and with an antibody for the alpha3/5 subunits of nAChR. In contrast, immunoreactivity of SK3 subunits was not found in enteric neurons. A co-immunoprecipitation assay with Triton X-100-soluble membrane fractions prepared from the ileum revealed an association of the SK2 subunit with the alpha3/5 subunits of nAChR. These results suggest that SK2 channels negatively regulate the excitation of enteric neurons via functional interactions with nAChRs. PMID:17953675

  9. Acetylcholine Acts through Nicotinic Receptors to Enhance the Firing Rate of a Subset of Hypocretin Neurons in the Mouse Hypothalamus through Distinct Presynaptic and Postsynaptic Mechanisms1,2

    PubMed Central

    Zhou, Wen-Liang; Gao, Xiao-Bing

    2015-01-01

    Abstract Hypocretin/orexin neurons regulate many behavioral functions, including addiction. Nicotine acts through nicotinic acetylcholine receptors (nAChRs) to alter firing rate of neurons throughout the brain, leading to addiction-related behaviors. While nAChRs are expressed in the hypothalamus and cholinergic fibers project to this structure, it is unclear how acetylcholine modulates the activity of hypocretin neurons. In this study, we stimulated hypocretin neurons in mouse brain slices with ACh in the presence of atropine to dissect presynaptic and postsynaptic modulation of these neurons through nAChRs. Approximately one-third of tested hypocretin neurons responded to pressure application of ACh (1 mM) with an increase in firing frequency. Stimulation of postsynaptic nAChRs with ACh or nicotine resulted in a highly variable inward current in approximately one-third of hypocretin neurons. In contrast, ACh or nicotine (1 µM) reliably decreased the frequency of miniature EPSCs (mEPSCs). Antagonism of nAChRs with mecamylamine also suppressed mEPSC frequency, suggesting that an endogenous, tonic activation of presynaptic nAChRs might be required for maintaining functional mEPSC frequency. Antagonism of heteromeric (α4β2) or homomeric (α7) nAChRs alone suppressed mEPSCs to a lesser extent. Finally, blocking internal calcium release reduced the frequency of mEPSCs, occluding the suppressive effect of presynaptic ACh. Taken together, these data provide a mechanism by which phasic ACh release enhances the firing of a subset of hypocretin neurons through postsynaptic nAChRs, but disrupts tonic, presynaptic nAChR-mediated glutamatergic inputs to the overall population of hypocretin neurons, potentially enhancing the signal-to-noise ratio during the response of the nAChR-positive subset of neurons. PMID:26322330

  10. Acetylcholinesterase-Fc Fusion Protein (AChE-Fc): A Novel Potential Organophosphate Bioscavenger with Extended Plasma Half-Life.

    PubMed

    Noy-Porat, Tal; Cohen, Ofer; Ehrlich, Sharon; Epstein, Eyal; Alcalay, Ron; Mazor, Ohad

    2015-08-19

    Acetylcholinesterase (AChE) is the physiological target of organophosphate nerve agent compounds. Currently, the development of a formulation for prophylactic administration of cholinesterases as bioscavengers in established risk situations of exposure to nerve agents is the incentive for many efforts. While cholinesterase bioscavengers were found to be highly effective in conferring protection against nerve agent exposure in animal models, their therapeutic use is complicated by short circulatory residence time. To create a bioscavenger with prolonged plasma half-life, compatible with biotechnological production and purification, a chimeric recombinant molecule of HuAChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein, expressed in cultured cells under optimized conditions, maintains its full enzymatic activity, at levels similar to those of the recombinant AChE enzyme. Thus, this novel fusion product retained its binding affinity toward BW284c5 and propidium, and its bioscavenging reactivity toward the organophosphate-AChE inhibitors sarin and VX. Furthermore, when administered to mice, AChE-Fc exhibits exceptional circulatory residence longevity (MRT of 6000 min), superior to any other known cholinesterase-based recombinant bioscavengers. Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents, and ease of production, AChE-Fc emerges as a promising next-generation organophosphate bioscavenger. PMID:26121420

  11. Neuronal Nicotinic Acetylcholine Receptors: Common Molecular Substrates of Nicotine and Alcohol Dependence

    PubMed Central

    Hendrickson, Linzy M.; Guildford, Melissa J.; Tapper, Andrew R.

    2013-01-01

    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 pre-clinical, 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. PMID:23641218

  12. Neurobiology of nAChRs and cognition: A mini review of Dr. Jerry J. Buccafusco's contributions over a 25 year career

    PubMed Central

    Terry, Alvin V.; Decker, Michael W.

    2011-01-01

    This review highlights some of the many contributions of the late Dr. Jerry J. Buccafusco to the neurobiology of nicotinic acetylcholine receptors (nAChRs) and cognition over a 25 year period. The article is written by two of Dr. Buccafusco's professional colleagues, one from academia and one from the pharmaceutical industry. While Dr. Buccafusco's expertise in the cholinergic field was extensive, his insights into the practical relevance of his work (with a long-term goal of formulating new drug development strategies) were unique, and a great asset to both the basic science community and pharmaceutical companies. In 1988, Dr. Buccafusco's laboratory was the first to report the cognitive enhancing action of low doses of nicotine in non-human primates. Since that time he studied a large number of novel pro-cognitive agents from several pharmacological classes in rodents as well as monkeys. Based on years of observing paradoxical effects of nicotinic ligands in vitro and in vivo, Dr. Buccafusco made the provocative argument that it might be possible to develop new chemical entities (with pro-cognitive actions) that have the ability to desensitize nAChRs without producing an antecedent agonist action. Some of his more recent work focused on development of single molecular entities that act on multiple CNS targets (including nAChRs) to enhance cognition, provide neuroprotection, and/or provide additional therapeutic actions (e.g., antipsychotic effects). Dr. Buccafusco's influence will live on in the work of the numerous graduate students, postdoctoral fellows, and junior faculty that he mentored over the years who now serve in prestigious positions throughout the world. PMID:21684265

  13. Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain.

    PubMed

    Wu, Jie; Liu, Qiang; Tang, Pei; Mikkelsen, Jens D; Shen, Jianxin; Whiteaker, Paul; Yakel, Jerrel L

    2016-07-01

    The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling. PMID:27179601

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

    SciTech Connect

    Buck, M.A.; Fraser, C.M. )

    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.

  15. Activation of nicotinic acetylcholine receptors increases the rate of fusion of cultured human myoblasts.

    PubMed Central

    Krause, R M; Hamann, M; Bader, C R; Liu, J H; Baroffio, A; Bernheim, L

    1995-01-01

    1. Fusion of myogenic cells is important for muscle growth and repair. The aim of this study was to examine the possible involvement of nicotinic acetylcholine receptors (nAChR) in the fusion process of myoblasts derived from postnatal human satellite cells. 2. Acetylcholine-activated currents (ACh currents) were characterized in pure preparations of freshly isolated satellite cells, proliferating myoblasts, myoblasts triggered to fuse and myotubes, using whole-cell and single-channel voltage clamp recordings. Also, the effect of cholinergic agonists on myoblast fusion was tested. 3. No nAChR were observed in freshly isolated satellite cells. nAChR were first observed in proliferating myoblasts, but ACh current densities increased markedly only just before fusion. At that time most mononucleated myoblasts had ACh current densities similar to those of myotubes. ACh channels had similar properties at all stages of myoblast maturation. 4. The fraction of myoblasts that did not fuse under fusion-promoting conditions had no ACh current and thus resembled freshly isolated satellite cells. 5. The rate of myoblast fusion was increased by carbachol, an effect antagonized by alpha-bungarotoxin, curare and decamethonium, but not by atropine, indicating that nAChR were involved. Even though a prolonged exposure to carbachol led to desensitization, a residual ACh current persisted after several days of exposure to the nicotinic agonist. 6. Our observations suggest that nAChR play a role in myoblast fusion and that part of this role is mediated by the flow of ions through open ACh channels. Images Figure 1 Figure 2 Figure 3 PMID:8788942

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

    PubMed

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

    2016-08-17

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

  17. Nicotinic α7 receptors enhance NMDA cognitive circuits in dorsolateral prefrontal cortex

    PubMed Central

    Yang, Yang; Paspalas, Constantinos D.; Jin, Lu E.; Picciotto, Marina R.; Arnsten, Amy F. T.; Wang, Min

    2013-01-01

    The cognitive function of the highly evolved dorsolateral prefrontal cortex (dlPFC) is greatly influenced by arousal state, and is gravely afflicted in disorders such as schizophrenia, where there are genetic insults in α7 nicotinic acetylcholine receptors (α7-nAChRs). A recent behavioral study indicates that ACh depletion from dlPFC markedly impairs working memory [Croxson PL, Kyriazis DA, Baxter MG (2011) Nat Neurosci 14(12):1510–1512]; however, little is known about how α7-nAChRs influence dlPFC cognitive circuits. Goldman-Rakic [Goldman-Rakic (1995) Neuron 14(3):477–485] discovered the circuit basis for working memory, whereby dlPFC pyramidal cells excite each other through glutamatergic NMDA receptor synapses to generate persistent network firing in the absence of sensory stimulation. Here we explore α7-nAChR localization and actions in primate dlPFC and find that they are enriched in glutamate network synapses, where they are essential for dlPFC persistent firing, with permissive effects on NMDA receptor actions. Blockade of α7-nAChRs markedly reduced, whereas low-dose stimulation selectively enhanced, neuronal representations of visual space. These findings in dlPFC contrast with the primary visual cortex, where nAChR blockade had no effect on neuronal firing [Herrero JL, et al. (2008) Nature 454(7208):1110–1114]. We additionally show that α7-nAChR stimulation is needed for NMDA actions, suggesting that it is key for the engagement of dlPFC circuits. As ACh is released in cortex during waking but not during deep sleep, these findings may explain how ACh shapes differing mental states during wakefulness vs. sleep. The results also explain why genetic insults to α7-nAChR would profoundly disrupt cognitive experience in patients with schizophrenia. PMID:23818597

  18. Allosteric effects of four stereoisomers of a fused indole ring system with 3H-N-methylscopolamine and acetylcholine at M1-M4 muscarinic receptors.

    PubMed

    Lazareno, S; Birdsall, B; Fukazawa, T; Gharagozloo, P; Hashimoto, T; Kuwano, H; Popham, A; Sugimoto, M; Birdsall, N J

    1999-01-01

    We previously demonstrated that brucine and some analogues allosterically enhance the affinity of ACh at muscarinic receptor subtypes M1, M3 or M4. Here we describe allosteric effects at human M1-M4 receptors of four stereoisomers of a pentacyclic structure containing features of the ring structure of brucine. All compounds inhibited 3H-NMS dissociation almost completely at all subtypes with slopes of 1, with similar affinity values at the 3H-NMS-occupied receptor to those estimated from equilibrium assays, consistent with the ternary complex allosteric model. Compound 1a showed positive cooperativity with H-NMS and small negative or neutral cooperativity with ACh at all subtypes. Its stereoisomer, 1b, showed strong negative cooperativity with both 3H-NMS and ACh across the subtypes. Compound 2a was positive with 3H-NMS at M2 and M4 receptors, neutral at M3 and negative at M1 receptors; it was negatively cooperative with ACh at all subtypes. Its stereoisomer, 2b, was neutral with 3H-NMS at M1 receptors and positive at the other subtypes; 2b was negatively cooperative with ACh at M1, M3 and M4 receptors but showed 3-fold positive cooperativity with ACh at M2 receptors. This latter result was confirmed with further 3H-NMS and 3H-ACh radioligand binding assays and with functional assays of ACh-stimulated 35S-GTPgammaS binding. These results provide the first well characterised instance of a positive enhancer of ACh at M2 receptors, and illustrate the difficulty of predicting such an effect. PMID:10069518

  19. Deposition of a-C:H films on a nanotrench pattern by bipolar PBII&D

    NASA Astrophysics Data System (ADS)

    Hirata, Yuki; Nakahara, Yuya; Nagato, Keisuke; Choi, Junho

    2016-06-01

    In this study, hydrogenated amorphous carbon (a-C:H) films were deposited on a nanotrench pattern (300 nm pitch, aspect ratio: 2.0) by bipolar-type plasma based ion implantation and deposition technique (bipolar PBII&D), and the effects of bipolar pulse on the film properties were investigated. Moreover, the behaviour of ions and radicals surrounding the nanotrench was analyzed to clarify the coating mechanism and properties of the a-C:H films on the nanotrench. Further, thermal nanoimprint lithography was carried out using the nanotrench pattern coated with a-C:H films as the mold, and the mold release properties were evaluated. All nanotrench surfaces were successfully coated with the a-C:H films, but the film thickness on the top, sidewall, and bottom surfaces of the trench were not uniform. The surface roughness of the a-C:H films was found to decrease at a higher positive voltage; this happens due to the higher electron temperature around the nanotrench because of the surface migration of plasma particles arrived on the trench. The effects of the negative voltage on the behaviour of ions and radicals near the sidewall of the nanotrench are quite similar to those near the microtrench reported previously (Park et al 2014 J. Phys. D: Appl. Phys. 47 335306). However, the positive pulse voltage was also found to affect the behaviour of ions and radicals near the sidewall surface. The incident angles of ions on the sidewall surface increased with the positive pulse voltage because the energy of incoming ions on the trench decreases with increasing positive voltage. Moreover, the incident ion flux on the sidewall is affected by the positive voltage history. Further, the radical flux decreases with increasing positive voltage. It can be concluded that a higher positive voltage at a lower negative voltage condition is good to obtain better film properties and higher film thickness on the sidewall surface. Pattern transfer properties for the nanoimprint formed by

  20. Structure and dynamics of the M3 muscarinic acetylcholine receptor

    SciTech Connect

    Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jürgen; Kobilka, Brian K.

    2012-03-01

    Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.

  1. Critical metabolic roles of β-cell M3 muscarinic acetylcholine receptors

    PubMed Central

    de Azua, Inigo Ruiz; Gautam, Dinesh; Jain, Shalini; Guettier, Jean-Marc; Wess, Jürgen

    2013-01-01

    Muscarinic acetylcholine (ACh) receptors (mAChRs; M1–M5) regulate the activity of an extraordinarily large number of important physiological processes. We and others previously demonstrated that pancreatic β-cells are endowed with M3 mAChRs which are linked to G proteins of the Gq family. The activation of these receptors by ACh or other muscarinic agonists leads to the augmentation of glucose-induced insulin release via multiple mechanisms. Interestingly, in humans, ACh acting on human β-cell mAChRs is released from adjacent α-cells which express both choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (vAChT), indicative of the presence of a non-neuronal cholinergic system in human pancreatic islets. In order to shed light on the physiological roles of β-cell M3 receptors, we recently generated and analyzed various mutant mouse models. Specifically, we carried out studies with mice which overexpressed M3 receptors or mutant M3 receptors in pancreatic β-cells or which selectively lacked M3 receptors or M3-receptor-associated proteins in pancreatic β-cells. Our findings indicate that β-cell M3 receptors play a key role in maintaining proper insulin release and whole body glucose homeostasis and that strategies aimed at enhancing signaling through β-cell M3 receptors may prove useful to improve β-cell function for the treatment of type 2 diabetes (T2D). PMID:22525375

  2. Animal models of myasthenia gravis: utility and limitations

    PubMed Central

    Mantegazza, Renato; Cordiglieri, Chiara; Consonni, Alessandra; Baggi, Fulvio

    2016-01-01

    Myasthenia gravis (MG) is a chronic autoimmune disease caused by the immune attack of the neuromuscular junction. Antibodies directed against the acetylcholine receptor (AChR) induce receptor degradation, complement cascade activation, and postsynaptic membrane destruction, resulting in functional reduction in AChR availability. Besides anti-AChR antibodies, other autoantibodies are known to play pathogenic roles in MG. The experimental autoimmune MG (EAMG) models have been of great help over the years in understanding the pathophysiological role of specific autoantibodies and T helper lymphocytes and in suggesting new therapies for prevention and modulation of the ongoing disease. EAMG can be induced in mice and rats of susceptible strains that show clinical symptoms mimicking the human disease. EAMG models are helpful for studying both the muscle and the immune compartments to evaluate new treatment perspectives. In this review, we concentrate on recent findings on EAMG models, focusing on their utility and limitations. PMID:27019601

  3. Blockade by 2,2',2''-tripyridine of the nicotinic acetylcholine receptor channels in embryonic Xenopus muscle cells.

    PubMed Central

    Hsu, K. S.; Fu, W. M.; Lin-Shiau, S. Y.

    1993-01-01

    1. The effects of 2,2',2''-tripyridine on the nicotinic acetylcholine (ACh) receptor channels were studied in the cultured myocytes of 1-day-old Xenopus embryos. 2. 2,2',2''-Tripyridine depressed the amplitude of iontophoretic ACh-induced current at a low frequency of 0.7 Hz stimulation and it not only decreased the initial responses but also enhanced the run-down of ACh-induced current at higher frequency stimulation of 7 Hz and 30 Hz. 3. Single ACh channel recordings showed that 2,2',2''-tripyridine decreased the channel conductance, the opening frequency and mean open time of both types of low- and high-conductance channels. 4. These results suggest that the blocking actions of 2,2',2''-tripyridine on ACh receptor channels in the skeletal muscle may contribute to the depression of the nerve-evoked contraction of the mouse diaphragm as reported previously. PMID:7693275

  4. [Autoantibodies in myasthenia gravis].

    PubMed

    Motomura, Masakatsu; Narita Masuda, Tomoko

    2013-04-01

    Myasthenia gravis (MG) is caused by the failure of neuromuscular transmission mediated by pathogenic autoantibodies (Abs). Generally, patients with MG are divided into 3 groups: (1) nicotinic acetylcholine receptor antibody-positive MG (AChR-MG: 80%), (2) muscle-specific receptor tyrosine kinase antibody-positive MG (MuSK-MG: 5-10%), which are AChR-associated transmembrane post-synaptic proteins involved in AChR aggregation, and (3) double-seronegative MG. In 2011, autoantibodies against low-density lipoprotein receptor-related protein 4 (Lrp4) were identified in Japanese MG patients, and thereafter, have been reported in Germany and USA. We developed a simple technique termed Gaussia luciferase immunoprecipitation (GLIP) for detecting the antibodies to Lrp4. Our results showed that 9 generalized MG patients out of 300 without AChR Ab were positive for Lrp4 antibodies. These antibodies inhibit the binding of Lrp4 to its ligand and predominantly belong to the IgG1 subclass. In other studies, Lrp4 Ab-positive sera inhibited agrin-induced aggregation of AChRs in cultured myotubes, suggesting a pathogenic role in the dysfunction of the neuromuscular endplate. Further understanding of the structure and function of neuromuscular junction (NMJ) through newly discovered autoantibodies may provide specific clinical information and treatment for MG. PMID:23568991

  5. Nicotinic receptor modulation to treat alcohol and drug dependence

    PubMed Central

    Rahman, Shafiqur; Engleman, Eric A.; Bell, Richard L.

    2015-01-01

    Alcohol and drug dependence are serious public health problems worldwide. The prevalence of alcohol and drug dependence in the United States and other parts of the world is significant. Given the limitations in the efficacy of current pharmacotherapies to treat these disorders, research in developing alternative pharmacotherapies continues. Preclinical and clinical evidence thus far has indicated that brain nicotinic acetylcholine receptors (nAChRs) are important pharmacological targets for the development of medications to treat alcohol and drug dependence. The nAChRs are a super family of ligand gated ion channels, and are expressed throughout the brain with twelve neuronal nAChR subunits (α2–α10 and β2–β4) identified. Here, we review preclinical and clinical evidence involving a number of nAChR ligands that target different nAChR subtypes in alcohol and nicotine addiction. The important ligands include cytisine, lobeline, mecamylamine, varenicline, sazetidine A and others that target α4β2* nAChR subtypes as small molecule modulators of the brain nicotinic cholinergic system are also discussed. Taken together, both preclinical and clinical data exist that support nAChR–based ligands as promising therapeutic agents for the treatment of alcohol and drug dependence. PMID:25642160

  6. Muscle aches

    MedlinePlus

    ... be done include: Complete blood count (CBC) Other blood tests to look at muscle enzymes (creatine kinase) and possibly a test for Lyme disease or a connective tissue disorder Physical therapy may be helpful.

  7. The importance of muscarinic receptors in domestic animal diseases and therapy: Current and future perspectives.

    PubMed

    Abraham, Getu

    2016-02-01

    This review provides an overview of the early and current literature including contributions that highlight the parasympathetic cholinergic receptor systems in domestic animal tissues. Muscarinic acetylcholine receptors (mAChRs) belong to the subfamily of G protein-coupled receptors and regulate many fundamental functions of the central and peripheral nervous systems and have been subject to research over at least 40 years. Nonetheless, there are few studies specifying mAChRs in domestic animal tissues. This review focuses on the pharmacology of muscarinic acetylcholine receptor (mAChR) system and its pathological as well as the therapeutic importance in organ systems of domestic animals. Illustration and discussion of recent advances in distribution, function, biochemistry and pharmacology of mAChRs are followed by summaries of the involvement of this family of receptors in cardiovascular, respiratory, neurological, gastrointestinal (GI) and urological diseases as well as in anaesthesia and toxicology. Specific functions of mAChRs are described in detail including subtype characterization, smooth muscle functions, signal transduction and regulation. Due to their wide tissue distribution, mAChRs have shown promise as targets for the treatment of some animal diseases such as equine recurrent airway obstruction, glaucoma, abnormalities of gastric acid secretion and GI disturbances including colic. PMID:26654844

  8. The immunomodulation of nicotinic acetylcholine receptor subunits in Zhikong scallop Chlamys farreri.

    PubMed

    Shi, Xiaowei; Zhou, Zhi; Wang, Lingling; Wang, Mengqiang; Shi, Shaoying; Wang, Zhen; Song, Linsheng

    2015-11-01

    Nicotinic acetylcholine receptor (nAChR), the best-studied ionotropic neuron receptor protein, is a key player in neuronal communication, and it has been reported to play an important role in immunomodulation of vertebrates. Although nAChRs have also been identified in most invertebrates, the knowledge about their immunomodulation is still limited. In the present study, two scallop nAChR genes were identified from Chlamys farreri (designed as CfnAChR1 and CfnAChR2), which encoded 384 and 443 amino acids, respectively. The conserved disulfide-linked cystines, ion selectivity residues and the hydrophobic gating residues (L251, V255 and V259) were identified in CfnAChR1 and CfnAChR2. The immunoreactivities of CfnAChR1 and CfnAChR2 were observed in all the tested scallop tissues, including adductor muscle, mantle, gill, hepatopancreas, kidney and gonad. After LPS (0.5 mg mL(-1)) stimulation, the expression of CfnAChR1 mRNA in haemocytes increased significantly by 9.83-fold (P < 0.05) and 12.93-fold (P < 0.05) at 3 h and 24 h, respectively. While the expression level of CfnAChR2 mRNA increased 43.94% at 12 h after LPS stimulation (P < 0.05). After TNF-α (50 ng mL(-1)) stimulation, the expression levels of CfnAChR1 and CfnAChR2 both increased significantly at 1 h, which were 21.33-fold (P < 0.05) and 2.44-fold (P < 0.05) of that in the PBS group, respectively. The results collectively indicated that the cholinergic nervous system in scallops could be activated by immune stimulations through CfnAChR1 and CfnAChR2, which function as the links between the cholinergic nervous system and immune system. PMID:26455648

  9. Menthol Binding and Inhibition of α7-Nicotinic Acetylcholine Receptors

    PubMed Central

    Ashoor, Abrar; Nordman, Jacob C.; Veltri, Daniel; Yang, Keun-Hang Susan; Al Kury, Lina; Shuba, Yaroslav; Mahgoub, Mohamed; Howarth, Frank C.; Sadek, Bassem; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-01-01

    Menthol is a common compound in pharmaceutical and commercial products and a popular additive to cigarettes. The molecular targets of menthol remain poorly defined. In this study we show an effect of menthol on the α7 subunit of the nicotinic acetylcholine (nACh) receptor function. Using a two-electrode voltage-clamp technique, menthol was found to reversibly inhibit α7-nACh receptors heterologously expressed in Xenopus oocytes. Inhibition by menthol was not dependent on the membrane potential and did not involve endogenous Ca2+-dependent Cl− channels, since menthol inhibition remained unchanged by intracellular injection of the Ca2+ chelator BAPTA and perfusion with Ca2+-free bathing solution containing Ba2+. Furthermore, increasing ACh concentrations did not reverse menthol inhibition and the specific binding of [125I] α-bungarotoxin was not attenuated by menthol. Studies of α7- nACh receptors endogenously expressed in neural cells demonstrate that menthol attenuates α7 mediated Ca2+ transients in the cell body and neurite. In conclusion, our results suggest that menthol inhibits α7-nACh receptors in a noncompetitive manner. PMID:23935840

  10. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    NASA Astrophysics Data System (ADS)

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin; Kramer, Richard H.; Trauner, Dirk

    2012-02-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels.

  11. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

    PubMed Central

    Tochitsky, Ivan; Banghart, Matthew R.; Mourot, Alexandre; Yao, Jennifer Z.; Gaub, Benjamin

    2016-01-01

    Advances in synthetic chemistry, structural biology, molecular modelling and molecular cloning have enabled the systematic functional manipulation of transmembrane proteins. By combining genetically manipulated proteins with light-sensitive ligands, innately ‘blind’ neurobiological receptors can be converted into photoreceptors, which allows them to be photoregulated with high spatiotemporal precision. Here, we present the optochemical control of neuronal nicotinic acetylcholine receptors (nAChRs) with photoswitchable tethered agonists and antagonists. Using structure-based design, we produced heteromeric α3β4 and α4β2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark. The generation of these engineered receptors should facilitate investigation of the physiological and pathological functions of neuronal nAChRs and open a general pathway to photosensitizing pentameric ligand-gated ion channels. PMID:22270644

  12. Specific Immunotherapy of Experimental Myasthenia Gravis by A Novel Mechanism

    PubMed Central

    Luo, Jie; Kuryatov, Alexander; Lindstrom, Jon

    2009-01-01

    Objective Myasthenia gravis (MG) and its animal model, experimental autoimmune myasthenia gravis (EAMG), are antibody-mediated autoimmune diseases, in which autoantibodies bind to and cause loss of muscle nicotinic acetylcholine receptors (AChRs) at the neuromuscular junction. To develop a specific immunotherapy of MG, we treated rats with ongoing EAMG by intraperitoneal injection of bacterially-expressed human muscle AChR constructs. Methods Rats with ongoing EAMG received intraperitoneal treatment with the constructs weekly for 5 weeks beginning after the acute phase. Autoantibody concentration, subclassification, and specificity were analyzed to address underlying therapeutic mechanism. Results EAMG was specifically suppressed by diverting autoantibody production away from pathologically relevant specificities directed at epitopes on the extracellular surface of muscle AChRs toward pathologically irrelevant epitopes on the cytoplasmic domain. A mixture of subunit cytoplasmic domains was more effective than a mixture containing both extracellular and cytoplasmic domains or than only the extracellular domain of α1 subunits. Interpretation Therapy using only cytoplasmic domains, which lack pathologically relevant epitopes, avoids the potential liability of boosting the pathological response. Use of a mixture of bacterially-expressed human muscle AChR cytoplasmic domains for antigen-specific immunosuppression of myasthenia gravis has the potential to be specific, robust, and safe. PMID:20437579

  13. A CHRNE frameshift mutation causes congenital myasthenic syndrome in young Jack Russell Terriers.

    PubMed

    Rinz, Caitlin J; Lennon, Vanda A; James, Fiona; Thoreson, James B; Tsai, Kate L; Starr-Moss, Alison N; Humphries, H Dale; Guo, Ling T; Palmer, Anthony C; Clark, Leigh Anne; Shelton, G Diane

    2015-12-01

    Congenital myasthenic syndromes (CMSs) are a group of rare genetic disorders of the neuromuscular junction resulting in structural or functional causes of fatigable weakness that usually begins early in life. Mutations in pre-synaptic, synaptic and post-synaptic proteins have been demonstrated in human cases, with more than half involving aberrations in nicotinic acetylcholine receptor (AChR) subunits. CMS was first recognized in dogs in 1974 as an autosomal recessive trait in Jack Russell Terriers (JRTs). A deficiency of junctional AChRs was demonstrated. Here we characterize a CMS in 2 contemporary cases of JRT littermates with classic clinical and electromyographic findings, and immunochemical confirmation of an approximately 90% reduction in AChR protein content. Loci encoding the 5 AChR subunits were evaluated using microsatellite markers, and CHRNB1 and CHRNE were identified as candidate genes. Sequences of the splice sites and exons of both genes revealed a single base insertion in exon 7 of CHRNE that predicts a frameshift mutation and a premature stop codon. We further demonstrated this pathogenic mutation in CHRNE in archival tissues from unrelated JRTs studied 34 years ago. PMID:26429099

  14. PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression

    PubMed Central

    Thomas, Jean-Luc; Ravel-Chapuis, Aymeric; Valente, Carmen; Corda, Daniela; Méjat, Alexandre

    2015-01-01

    Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway. PMID:26416879

  15. PAK1 and CtBP1 Regulate the Coupling of Neuronal Activity to Muscle Chromatin and Gene Expression.

    PubMed

    Thomas, Jean-Luc; Moncollin, Vincent; Ravel-Chapuis, Aymeric; Valente, Carmen; Corda, Daniela; Méjat, Alexandre; Schaeffer, Laurent

    2015-12-01

    Acetylcholine receptor (AChR) expression in innervated muscle is limited to the synaptic region. Neuron-induced electrical activity participates in this compartmentalization by promoting the repression of AChR expression in the extrasynaptic regions. Here, we show that the corepressor CtBP1 (C-terminal binding protein 1) is present on the myogenin promoter together with repressive histone marks. shRNA-mediated downregulation of CtBP1 expression is sufficient to derepress myogenin and AChR expression in innervated muscle. Upon denervation, CtBP1 is displaced from the myogenin promoter and relocates to the cytoplasm, while repressive histone marks are replaced by activating ones concomitantly to the activation of myogenin expression. We also observed that upon denervation the p21-activated kinase 1 (PAK1) expression is upregulated, suggesting that phosphorylation by PAK1 may be involved in the relocation of CtBP1. Indeed, preventing CtBP1 Ser158 phosphorylation induces CtBP1 accumulation in the nuclei and abrogates the activation of myogenin and AChR expression. Altogether, these findings reveal a molecular mechanism to account for the coordinated control of chromatin modifications and muscle gene expression by presynaptic neurons via a PAK1/CtBP1 pathway. PMID:26416879

  16. Deposition of a-C:H films on UHMWPE substrate and its wear-resistance

    NASA Astrophysics Data System (ADS)

    Xie, Dong; Liu, Hengjun; Deng, Xingrui; Leng, Y. X.; Huang, Nan

    2009-10-01

    In prosthetic hip replacements, ultrahigh molecular weight polyethylene (UHMWPE) wear debris is identified as the main factor limiting the lifetime of the artificial joints. Especially UHMWPE debris from the joint can induce tissue reactions and bone resorption that may lead to the joint loosening. The diamond like carbon (DLC) film has attracted a great deal of interest in recent years mainly because of its excellent tribological property, biocompatibility and chemically inert property. In order to improve the wear-resistance of UHMWPE, a-C:H films were deposited on UHMWPE substrate by electron cyclotron resonance microwave plasma chemical vapor deposition (ECR-PECVD) technology. During deposition, the working gases were argon and acetylene, the microwave power was set to 800 W, the biased pulsed voltage was set to -200 V (frequency 15 kHz, duty ratio 20%), the pressure in vacuum chamber was set to 0.5 Pa, and the process time was 60 min. The films were analysed by X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, nano-indentation, anti-scratch and wear test. The results showed that a typical amorphous hydrogenated carbon (a-C:H) film was successfully deposited on UHMWPE with thickness up to 2 μm. The nano-hardness of the UHMWPE coated with a-C:H films, measured at an applied load of 200 μN, was increased from 10 MPa (untreated UHMWPE) to 139 MPa. The wear test was carried out using a ball (Ø 6 mm, SiC) on disk tribometer with an applied load of 1 N for 10000 cycles, and the results showed a reduction of worn cross-sectional area from 193 μm 2 of untreated UHMWPE to 26 μm 2 of DLC coated sample. In addition the influence of argon/acetylene gas flow ratio on the growth of a-C:H films was studied.

  17. The stabilization of Au NP-AChE nanocomposites by biosilica encapsulation for the development of a thiocholine biosensor.

    PubMed

    Buiculescu, Raluca; Chaniotakis, Nikos A

    2012-08-01

    We report on the construction of an amperometric biosensor based on the immobilization of the enzyme acetylcholinesterase (AChE) onto gold nanoparticles (Au NPs). The active enzyme is covalently bound directly onto the surface of the Au NPs via a thiol bond. This immobilization provides increased stability and high electron-transfer between the colloidal Au NPs, the catalyst and the transducer surface. To further increase the biosensor stability by protecting the enzyme from denaturation and protease attack, a layer of biosilica was grown around the Au NP enzyme nanocomposite. All steps, i.e., the conjugation of the enzyme to the gold nanoparticles and the encapsulation into biosilica, are monitored and confirmed by ATR-FT-IR spectroscopy. The stabilizing effect of the entrapment was evaluated amperometrically, while the operation of the biosensor was monitored over a period of 4 months. The initial sensitivity of the biosensor was calculated to be 27.58 nA mM(-1) with a linear response to the concentration of the substrate in the range from 0.04 to 0.4 mM. It is thus shown that the biosilica nanocomposites doped with Au NPs-AChE conjugates create a system that provides both signal mediation and significant enzyme stabilization over the existing AChE biosensor. The biosensor had retained all its activity at the end of the 4 months, compared with the normal AChE biosensor whose activity reached 50% after only 42 days of operation. PMID:22421347

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

    SciTech Connect

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

    1987-05-01

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

  19. Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells.

    PubMed

    Xu, Man; Bi, Xueyuan; He, Xi; Yu, Xiaojiang; Zhao, Ming; Zang, Weijin

    2016-05-18

    The mitochondrial unfolded protein response (UPR(mt)) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR(mt) in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR(mt) in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR(mt) and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR(mt) might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR. PMID:27111378

  20. Does Your Patient’s Urine Turns Dark? Alkaptonuria and Low Back Ache: A Literature Review

    PubMed Central

    Kanniyan, Kalaivanan; Pathak, Aditya C; Dhammi, Ish Kumar; Jain, Anil Kumar

    2014-01-01

    Introduction: Alkaptonuria is a very rare inborn error of amino acid metabolism due to deficient homogentisic acid (HGA) oxidase enzyme leading to accumulation of HGA in plasma, cartilage, other tissues of human body and its excretion in urine. It has both systemic and peripheral signs and symptoms. Though low back is a common symptom of alkaptonuria but, in the absence of ochronosis it is rare. Alkaptonuria itself is very rare occurrence with no specific treatment option available to reverse the effect as yet. Case Report: A 38-year-old male, embroidery worker presented with chronic low back ache with history of staining of clothes in infancy. Later on laboratory and the radiological investigation patient was diagnosed to have alkaptonuria without ochronosis. No other systemic manifestation was present. Patient was treated conservatively and responded well. Conclusion: Though alkaptonuria is a very rare disease, and the occurrence of low back-ache in absence of ochronosis is much rarer. One must be aware of this inborn error of metabolism. Early diagnosis though being “diagnosis of exclusion” for low back-ache, high index of suspicion is advantageous as symptomatic treatment of the alkaptonuria can be initiated and evaluation of other systemic organs can be done in early stages itself. PMID:27298997

  1. Novel multipotent AChEI-CCB attenuates hyperhomocysteinemia-induced memory deficits and Neuropathologies in rats.

    PubMed

    Xia, Yiyuan; Liu, Rong; Chen, Rong; Tian, Qing; Zeng, Kuan; Hu, Jichang; Liu, Xinghua; Wang, Qun; Wang, Peng; Wang, Xiao-Chuan; Wang, Jian-Zhi

    2014-01-01

    Alzheimer's disease (AD) has multiple etiopathogenic factors, yet the definitive cause remains unclear and the therapeutic strategies have been elusive. Combination therapy, as one of the promising treatments, has been studied for years and may exert synergistic beneficial effects on AD through polytherapeutic targets. In this study, we tested the effects of a synthesized juxtaposition (named SCR1693) composed of an acetylcholinesterase inhibitor (AChEI) and a calcium channel blocker (CCB) on the hyperhomocysteinemia (HHcy)-induced AD rat model, and found that SCR1693 remarkably improved the HHcy-induced memory deficits and preserved dendrite morphologies as well as spine density by upregulating synapse-associated proteins PSD95 and synapsin-1. In addition, SCR1693 attenuated HHcy-induced tau hyperphosphorylation at multiple AD-associated sites by regulating the activity of protein phosphatase-2A and glycogen synthase kinase-3β. Furthermore, SCR1693 was more effective than individual administration of both donepezil and nilvadipine which were used as AChEI and CCB, respectively, in the clinical practice. In conclusion, our data suggest that the polytherapeutic targeting juxtaposition SCR1693 (AChEI-CCB) is a promising therapeutic candidate for AD. PMID:25024319

  2. Neuromuscular therapeutics by RNA-targeted suppression of ACHE gene expression.

    PubMed

    Dori, Amir; Soreq, Hermona

    2006-10-01

    RNA-targeted therapeutics offers inherent advantages over small molecule drugs wherever one out of several splice variant enzymes should be inhibited. Here, we report the use of Monarsen, a 20-mer acetylcholinesterase-targeted antisense agent with three 3'-2'o-methyl-protected nucleotides, for selectively attenuating the stress-induced accumulation of the normally rare, soluble "readthrough" acetylcholinesterase variant AChE-R. Acetylcholine hydrolysis by AChE-R may cause muscle fatigue and moreover, limit the cholinergic anti-inflammatory blockade, yielding inflammation-associated pathology. Specific AChE-R targeting by Monarsen was achieved in cultured cells, experimental animals, and patient volunteers. In rats with experimental autoimmune myasthenia gravis, oral delivery of Monarsen improved muscle action potential in a lower dose regimen (nanomolar versus micromolar), rapid and prolonged manner (up to 72 h versus 2-4 h) as compared with the currently used small molecule anticholinesterases. In central nervous system neurons of both rats and cynomolgus monkeys, systematic Monarsen treatment further suppressed the levels of the proinflammatory cytokines interleukin-1 (IL-1) and IL-6. Toxicology testing and ongoing clinical trials support the notion that Monarsen treatment would offer considerable advantages over conventional cholinesterase inhibitors with respect to dosing, specificity, side effects profile, and duration of efficacy, while raising some open questions regarding its detailed mechanism of action. PMID:17145929

  3. Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new opportunities.

    PubMed

    Moaddel, Ruin; Jozwiak, Krzysztof; Wainer, Irving W

    2007-09-01

    Allosteric, non-competitive inhibitors (NCIs) of neuronal nicotinic acetylcholine receptors (nAChRs) have been shown to produce a wide variety of clinically relevant responses. Many of the observed effects are desired as the nAChR is the therapeutic target, while others are undesired consequences due to off-target binding at the nAChR. Thus, the determination of whether or not a lead drug candidate is an NCI should play an important role