Dissertations / Theses on the topic 'Acetylcholine'
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1
Tornoe, Calilla. "Nicotinic acetylcholine receptors in nematodes." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.363452.
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Bird, Martin Charles. "Immunochemistry of the acetylcholine receptor." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.370159.
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1. Anti-Torpedo marmorata AChR antibody fragments (Fab and F(ab')2) have been prepared from sheep immunised with Torpedo AChR, and exhibiting experimental autoimmune myasthenia gravis. The antibody fragments were labelled with 125I with the retention of antigen binding capacity. Labelled and unlabelled antibody fragments were used to study the antigenicity of soluble and membrane-bound AChR, Anti-receptor antibodies were found to reduce (?-toxin binding to the AChR This was demonstrated to be caused by steric hindrance rather than by direct blockade of the toxin binding site, or by antigenic modulation of the receptor. Removal of carbohydrate residues from the AChR resulted in no decrease in antibody binding, implying that carbohydrate made no direct contribution to the antigenicity of the receptor. Denaturation of the AChR resulted in a decrease in anti-receptor antibody binding of between 60 and 84%. Thus antibodies were directed mainly at conformation-dependent sites on the receptor. Substantial differences between the antigenic sites of soluble and membrane-bound receptors were found. 2. The contribution of antibody-mediated muscle cell lysis in the pathogenesis of myasthenia gravis has been studied, using a novel in vitro cytotoxicity assay based on 3H-carnitine uptake and release by muscle cells in culture. Cytolytic activity toward both chick and human embryonic muscle cell cultures was demonstrated in over 30% of myasthenic sera, suggesting that myolysis may be a major mechanism for AChR loss in myasthenia gravis. Heat-inactivation of the sera abolished their lytic activity, and it was not fully restored by the addition of guinea pig complement. Myolysis may be caused by antibodies other than anti-AChR antibodies present in myasthenic sera.
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Pagan, Augustine J. IV. "Heterosandwich assay of nicotinic acetylcholine receptors." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3815.
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Using the technology afforded by Winschel et al., cyclen-1, a high affinity, strong complexation agent for 8-hydroxypyrene-1,3,6 trisulfonate and derivatives, a new assay has been developed for fluorescently labeling proteins of interest (POIs). Ligation of the endogenous ligand for nicotinic acetylcholine receptors (nAChRs), acetylcholine, using click chemistry afforded the triazole derivative of an alkynyl-acylcholine (compound 1) with 8-azidopyrene-1,3,6 trisulfonate (compound 2). Liposomes encapsulated with Rhodamine B were used to strengthen the initial fluorophore response of compound 2, using an anchored form of cyclen-1 complex. Using a palmitoyl tail as the lipophilic moiety for liposomal amplification, the subsequent response has a fluorophore ratio of up to 1:1 million, compound 2:Rhodamine B molecules. in vitro assay using compound 2 and cyclen-1 anchored liposomes with HEK-293 cells produced a positive binding response, allowing brightly colored fluorescent images of nAChRs upon the cellular membrane. A control for nAChR binding was performed using a co-culture of HEK-293 and endothelial cell lines. Control experiments show compound 2 and liposomes weak binding endothelial cells, however, this could be do to accumulation from another mechanism, more work is necessary to prove whether or not this is correct.
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Burdon, Drew. "Cell growth regulation by muscarinic acetylcholine receptors." Thesis, University of Leicester, 2002. http://hdl.handle.net/2381/29932.
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The growth response of Chinese hamster ovary (CHO) cells to activation of recombinantly expressed G protein-coupled muscarinic M2 or M3 acetylcholine (ACh) receptors has been assessed. Activation of these receptors leads to divergent growth responses: M2 ACh receptor activation causes an increase in DNA synthesis, whereas M3 ACh receptor activation causes a dramatic inhibition of DNA synthesis. The M3 ACh receptor-mediated growth inhibition has been characterised, and shown to comprise a G1-phase cell cycle arrest, involving an increase in p21Cip1/Waf1 protein expression. Further, a receptor-mediated increase in p21Cip1/Waf1 association with cyclin-dependent kinase 2 (CDK2) leads to a decrease in CDK2 activity and an accumulation of hypophosphorylated retinoblastoma protein (pRb). The increase p21Cip1/Waf1 expression is due at least in part to an increase p21Cip1/Waf1 mRNA although no receptor-mediated change in candidate transcription factor activities could be detected. Extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation profiles suggested two alternative hypotheses to account for the receptor-mediated growth arrest. However, pharmacological and biochemical data demonstrate that ERK, JNK and p38 MAPK are not involved in the growth regulation, whilst inhibition of PKC partially ablates the growth arrest. Data demonstrate that both M3 ACh receptor-mediated ERK and JNK activities may be dependent on liberated G-protein bg subunits, whilst the growth arrest is not perturbed by bg subunit sequestration. Data presented reveal a MAPK-independent mechanism of growth regulation that may involve coping of the M3 ACh receptor to heterotrimeric G-protein families other than Gq/11.
5
Komourian, Jacques. "Alpha-bungarotoxin sensitive neuronal nicotinic acetylcholine receptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29731.pdf.
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Kommalage, Mahinda. "Spinal Acetylcholine Release : Mechanisms and Receptor Involvement." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-5931.
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Schött, Pär A. "Hippocampal galanin and acetylcholine in spatial learning /." Stockholm, 2000. http://diss.kib.ki.se/2000/91-628-4137-8/.
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Spalding, Tracy Anne. "Structural studies on the muscarinic acetylcholine receptor." Thesis, University College London (University of London), 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315419.
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Walsh, Susan. "Search for nicotinic acetylcholine receptors on lymphocytes." Thesis, University of Bath, 1989. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760593.
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10
Lotwick, Helen Sylvia. "Anti-(acetylcholine receptor) antibodies in myasthenia gravis." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.351788.
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Levels of anti-(AChR) antibodies were determined in serial serum samples from 14 myasthenic patients over a period of several months, using detergent-solubilized muscle extracts of junctional rat AChR, extra-junctional rat AChR and human adult AChR as antigens. Anti-(AChR) antibody titres obtained using human adult AChR were always higher than those obtained using extra-junctional rat AChR, which were, in turn, always higher than those obtained using junctional rat AChR. Ihe ratios of antibody titres obtained by using the different antigens varied between patients, but were constant for an individual over the period of study. Complementary evidence for the same phenamenon was obtained by other experiments in which an excess of each serum was used to precipitate limited amounts of AChR from muscle extracts. The results obtained by conbining myasthenic sera argue against the suggestion that incomplete precipitation of receptor by certain sera is caused by the absence of particular antibody sub-populations. An alternative explanation, that sera precipitating low amounts of AChR contain toxin-releasing antibodies, is supported by direct measuranents of antibody-mediated toxin loss. The hypothesis that embryonic AChR may constitute the autoimmunogen in MG vas investigated by comparing the interaction of human foetal AChR with BGT and anti-(AChR) antibodies against that established for adult human AChR. Tissue sections and teased muscle fibres fron human adult and foetal muscle were compared immunohistochemically. Detergent extracts of adult and foetal AChRs were canpared in their interaction with radiolabelled BGT by kinetic measurements involving determination of association, dissociation and equilibrium binding constants. AChR was isolated and partially purified frcm human adult and foetal muscle, and their binding to anti-(AChR) antibodies in myasthenic sera and IgG were compared. No significant difference was observed between the binding characteristics of the two receptor types, indicating the absence (at least in 14 - 22 week old foetuses) of ligand binding or antigenic sites unique to foetal AChR.
11
Madziva, Michael Taurai. "Mechanisms of M4 muscarinic acetylcholine receptor endocytosis." Thesis, University of Cambridge, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.619733.
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Demmerly, Arianna L. "Mechanisms of modulation of nicotinic acetylcholine receptors." Thesis, University of Alaska Fairbanks, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10244902.
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Inappropriate expression of nicotinic acetylcholine receptors in the central nervous system is associated with nicotine addiction, Alzheimer’s disease, Parkinson’s disease and other disorders. Modulators (drugs) have the potential to restore circuit properties that arise from inappropriate expression of nicotinic receptor’s. Compounds that interact with allosteric sites have a distinct advantage over agonists and partial agonists, in that, they retain normal activation patterns by allowing binding of the endogenous ligand. Positive allosteric modulators boost the receptors ability to respond to agonist. Studies of these modulators constitute a first step toward the identification and development of better compounds that minimize signaling errors at cholinergic synapses. We have used single molecule methods to investigate the action of a novel positive allosteric modulator, desformylflustrabromine (dFBr), on nicotinic receptors. Our studies were focused on the α4β2 subtype of nicotinic receptors in the brain. These receptors exist in two forms with low sensitivity (α42β23) or, alternatively, high sensitivity (α42β23) to agonist. Our experiments allowed us to develop detailed gating models for high and low sensitivity receptors, as well as gain new insights regarding the mechanisms that underlie potentiation by allosteric modulators. We found that dFBr potentiates low sensitivity receptors by destabilizing desensitized states of the receptor. In contrast, potentiation of high sensitivity receptors arises from a synchronization of openings following an application of agonist due to an increase in the opening rate. Based on our results we now have a better understanding of the advantages of dFBr on high and low sensitivity receptors.
13
Abramsson, Mia. "Production and characterization of Acetylcholine Binding Protein." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-355060.
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Chatzidaki, A. "Pharmacological characterisation of neuronal nicotinic acetylcholine receptors." Thesis, University College London (University of London), 2015. http://discovery.ucl.ac.uk/1473233/.
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Nicotinic acetylcholine receptors (nAChRs) are the targets for the endogenous neurotransmitter acetylcholine and represent a diverse family of ligand-gated ion channels. They are expressed in the neuromuscular junction, the peripheral nervous system and the central nervous system. In the brain, the most prevalent subtypes are the heteromeric α4β2 and homomeric α7 nAChRs. Neuronal nAChRs are implicated in numerous physiological and pathophysiological functions and are therefore important targets for therapeutic drug discovery for conditions such as Alzheimer’s disease, schizophrenia and tobacco addiction. This thesis aims to further our understanding of the pharmacological and molecular characteristics of neuronal nAChRs. Acetylcholine activates nAChRs by binding at an extracellular orthosteric site. Previous studies have described several ligands that potentiate agonist-evoked responses by binding to an allosteric site of the α7 nAChRs that is distinct from the acetylcholine binding site. These ligands, termed positive allosteric modulators (PAMs) can be described as type I, when they have little or no effect on desensitisation, or type II, when they dramatically slow down the fast desensitisation kinetics of the α7 nAChR subtype. Here, a novel series of α7-selective PAMs with a range of effects on receptor desensitisation is described, using recombinant human receptors. This series consists of PAMs with type I and type II profiles, in addition to PAMs with intermediate properties on desensitisation, therefore increasing the nAChR pharmacological toolbox. Furthermore, the effect of a number of mutations on the pharmacological properties of the receptor is investigated. Three point mutations, two in the transmembrane domain (L247T and M260L) and one in the N-terminal domain (W54A), are shown to have the ability to convert PAMs into agonists. Moreover, the M260L mutation displays this property only with PAMs that have a significant effect on receptor desensitisation. These observations can provide insights into the role of these residues on receptor gating and desensitisation. In addition to the studies on recombinant receptors, the expression and functional properties of nAChRs in neurons derived from human induced pluripotent stem cells (iPSC) is examined. The iPSC-derived neurons represent a potentially valuable tool for the characterisation of neuronal receptors and ion channels in a native environment.
15
Aslanoglou, Despoina. "Ligand regulation of muscarinic acetylcholine receptor organisation." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/7048/.
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Muscarinic acetylcholine receptors (M1-M5) belong to the class A family of transmembrane G protein coupled receptors (GPCRs) and mediate various signalling processes. M1, M3 and M5 predominantly couple to Gq and promote intracellular calcium ion release from the endoplasmic reticulum. M2 and M4 preferentially couple Gi inhibiting adenylyl cyclase activity to thus decrease cAMP production and acting to regulate various ion channels. There is growing evidence that many GPCRs can exist as dimers or higher-order oligomers (Milligan, 2013) and muscarinic receptors are no exception (Alvarez-Curto et al., 2010). Herein, combinations of homomers and heteromers of co-expressed human M2 (hM2WT) and a RASSL (Receptor Activated Solely by Synthetic Ligand) form of the human M3 receptor (hM3RASSL) (Alvarez-Curto et al., 2011) were demonstrated to occur using N-terminal SNAP and CLIP tags in combination with homogeneous time resolved FRET (htrFRET). Stable Flp-In™ T-REx™ 293 cell lines able to inducibly express each of these receptor forms upon addition of doxycycline, and a cell line able to express both the hM3RASSL constitutively and hM2WT in a doxycycline inducible manner were generated. In these cells both hM3RASSL and hM2WT were detected after treatment with different concentrations of doxycycline via Western blots using tag-specific antibodies. Radioligand binding using [3H]-QNB indicated that similar amounts of hM2WT and hM3RASSL were expressed following induction with 5 ng.ml-1 doxycycline in the cells co-expressing the two receptors. Expression of the receptors was observed at the surface of live cells following labelling of the expressed receptors with SNAP and CLIP-specific cell impermeant substrates. Following induction with doxycycline each of hM2WT and hM3RASSL homomers and hM2WT-hM3RASSL heteromers were identified. Detection of oligomers was achieved following co-labelling with htrFRET-compatible substrates. Occupancy of hM2WT-hM3RASSL heteromers with the hM2WT agonist carbachol resulted in a marked, time and concentration-dependent decrease in detected heteromers and a concomitant, concentration-dependent increase in hM2WT homomers. The dynamics of interchange between heteromers and homomers was investigated by using a multiplex labelling approach and htrFRET. This method involved labelling with one energy donor and two energy acceptors capable of emitting at distinct wavelengths. Results confirmed the hM2WT-hM3RASSL heteromer to hM2WT homomer transition upon selective carbachol-mediated activation of hM2WT. A small increase in the hM3RASSL homomer was detected upon activation of the hM3RASSL with the selective agonist clozapine-N-oxide, but this was only observed in the absence of heteromers. Despite the presence of hM2WT-hM3RASSL heteromers the functional pharmacology of hM2WT and hM3RASSL receptor specific agonists was largely unaltered.
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Rapier, Catherine Margaret. "Characterisation of mammalian central nicotinic acetylcholine receptors." Thesis, University of Bath, 1986. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.374614.
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Day, Toni. "Non-classical actions of acetylcholinesterase and related peptides on the in vitro hippocampus." Thesis, University of Oxford, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.249172.
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Iarriccio, Silva Laura. "Allosteric interactions at the M3 muscarinic acetylcholine receptor." Doctoral thesis, Universitat Politècnica de Catalunya, 2008. http://hdl.handle.net/10803/6469.
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The extracellular loops of muscarinic acetylcholine receptors are predicted to play a role in the binding and regulation of allosteric modulators. Furthermore, the sequence of the five subtypes of muscarinic receptors shows a large degree of diversity in this region. M3 receptor mutants, K523E, D518K and N132G, in which the substituted residues were those corresponding to the M1 subtype, were studied. As the amino acids in positions 518 and 523 are charged, the uncharged mutants, K523Q and D518N, were also created in order to observe any possible effect of charge. One question examined is whether these mutations changed the binding of orthosteric and allosteric ligands, generating a M1 receptor phenotype.Radioligand binding experiments revealed that one mutant, K523E, had a profound potentiating effect on the binding of prototypical modulators like gallamine, strychnine, brucine and N-chloromethylbrucine, but had minimal effects on the binding of a number of orthosteric ligands, including [3H]N-methylscopolamine ([3H]NMS) and acetylcholine (ACh). The increase in affinity was found at both the unoccupied and [3H]NMS-occupied receptors, with up to 70 fold increases in affinity being observed. Switches from negative to positive cooperativity for some strychnine-related compounds were found.
At K523E, the affinities of the strychnine-related ligands were also increased up to 160 fold at the receptor-ACh complex, with up to 35 fold positive cooperativity being observed. Positive cooperativity of this magnitude is the highest that has been reported for M3 receptors.
The dramatic changes in cooperativities and affinities of allosteric ligands at K523E did not result in generation of the M1 phenotype. The K523Q data suggest that the large changes in K523E result from the introduction of the negatively charged glutamate residue and not the loss of the positively charged lysine. The effect of K523E seems to be solely on the binding of allosteric ligands and the transmission of the effects of their binding to the orthosteric site.
For the ligands acting at the gallamine site, all the effects of the allosteric modulators on ACh binding have been reproduced in functional studies, indicating that the allosteric modulation, seen in binding, is transmitted to the cellular response. A novel and unexpected finding is that WIN62,577 is an allosteric agonist at M3 muscarinic receptors and at K523E and N132G. The study also revealed that nanomolar concentrations of ACh may be present in assays of muscarinic receptor function and may give misleading interpretations of data. These artefacts were removed by preincubation with acetylcholinesterase, a control not previously used in functional studies of muscarinic receptors.
The sensitivity of the binding of both orthosteric and allosteric ligands to the composition of the binding assay buffer has also been investigated in detail. In a phosphate buffer of low ionic strength (PB) the affinity constants of all the compounds studied, both orthosteric and allosteric, were increased, relative to a Hepes buffer of higher ionic strength, except for WIN 62,577, an allosteric ligand which binds to a different allosteric site from the prototypical modulators, and SVT-40776 a new M3 selective antagonist, indicating their different modes of binding. Cooperativities have also been switched from negative to positive by changing buffer.
The two factors affecting the allosteric binding parameters of M3 receptors, PB and the mutation K523E, mutually potentiate each others effects. We have been able to obtain up to 10,000 fold changes in the affinity at the unoccupied receptor and 6400 fold increases in affinity at the ACh occupied receptor.
The possible location of K523, relative to other residues on the external loops of muscarinic receptors shown to be important for the binding of allosteric ligands, has been explored using different models based on the X-ray structures of rhodopsin and the â2 adrenergic receptor.
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Minces, Victor. "Acetylcholine, brain activation, and processing of temporal stimuli." Diss., [La Jolla] : University of California, San Diego, 2010. http://wwwlib.umi.com/cr/fullcit?p3408026.
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Thesis (Ph. D.)--University of California, San Diego, 2010.Title from first page of PDF file (viewed June 22, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 201-214).
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Rankin, Saffron Emily. "Lipid-protein interactions and nicotinic acetylcholine receptor function." Thesis, University of Oxford, 1996. https://ora.ox.ac.uk/objects/uuid:3deca85b-9f09-4f72-9db3-e34851e10542.
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The effect of bilayer composition, specifically the presence of cholesterol, upon the function of the reconstituted nicotinic acetylcholine receptor (nAcChoR) was investigated using stopped-flow fluorescence. The nAcChoR was purified and reconstituted from the electroplaques of Torpedo nobiliana, using affinity column chromatography, into bilayers of defined composition and the function of each sample assessed and compared with those of the native receptor. Investigation of the effect of bilayer composition upon the kinetics of agonist binding to the nAcChoR, using the fluorescent acetylcholine analogue, Dns-C6-Cho, established that the receptor pre-existed in equilibrium between the resting and two desensitised states. However, Dns-C6-Cho inhibited channel gating at high concentrations and another fluorescent probe was sought. The kinetics of carbachol induced nAcChoR conformational changes, reported by ethidium bromide (a non-competitive inhibitor) fluorescence, in native membranes were characterised and an assay developed to investigate whether cholesterol mediated rapid conformational changes in reconstituted samples. It was found that ethidium bromide reported on the carbachol-induced development of the fast desensitised state from the open channel state, and that this conformational change was sensitive to changes in bilayer composition. The onset of fast desensitisation from the open channel state was not observed when the receptor was reconstituted into DOPC or DOPC-DOPA bilayers. However, increasing the cholesterol content in these bilayers increased the amplitude of the component reporting this conformational change, while the observed rate at which it occurred was independent of bilayer cholesterol content. This result agrees with the suggestion that cholesterol facilitates channel opening and the onset of fast desensitisation by binding to specific sites on the nAcChoR and that these must be occupied for a functionally viable receptor (Jones and McNamee, 1988).
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Crawford, Nicola. "Nicotinic acetylcholine receptors and their interaction with Aβ₁₋₄₂." Thesis, University of Edinburgh, 2006. http://hdl.handle.net/1842/29079.
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Radioligand binding was used to characterise the α4β2 nAChR ([3H]-epibatidine and [3H]-cytisine) and α7 nAChR ([3H]-methyllycaconitine ([3H]-MLA) and [3H]-αBungarotoxin ([3H]-αBgTx)) in rat and mouse brain tissue and in SH-EP1 cell lines overexpressing human forms of the α4β2 or α7 nAChRs. No species difference in ligand affinities were observed for the α4β2 nAChR. In contrast, nicotinic agonists exhibited significantly higher affinity (~100 fold) for human α7 nACRs compared to their rat counterparts with no change in antagonist affinity. Interestingly, evaluation of [3H]-MLA and [3H]-αBgTx binding indicated the latter ligand bound to a restricted number of sites on the α7 nAChR. Furthermore, neither human nor rat Aβ1-42 inhibited [3G[-cytisine or [3H]-MLA binding to nAChRs. In parallel to behavioural studies, these binding assays were also employed to assess nAChR pharmacology in transgenic α7 knockout mice. With no alteration in α4β2 nAChR pharmacology, the deficit in sustained attention exhibited by these α7-KO mice is probably due to loss of the α7 nAChR. Finally, a series of studies were performed to examine the functional interaction between Aβ1-42 and nAChRs. (-)Nicotine evoked changes in calcium flux or membrane potential in SH-EP1-hα4β2 cells were not inhibited by soluble or insoluble human Aβ1-42. Even whole cell patch clamp analysis of single cells showed no direct interaction between the α4β2 nAChR and Aβ1-42. The examination of the functional interaction between α7 nAChRs and Aβ1-42 using whole cell patch clamp or fluorescence based assays was compromised by a lack of consistent expression of functional human α7 nAChRs in the SH-EP1 cell line. In addition, neither human α4β2 or α7 nAChRs co-immunoprecipitated with human Aβ1-42.
22
Afar, Ronith. "Regulation and function of neuronal nicotinic acetylcholine receptors." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41288.
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Two major subtypes of nicotinic acetylcholine receptors (nAChRs) have been identified in the nervous system. One site is bound by $ alpha$-bungarotoxin ($ alpha$-BGT), while the other has a higher affinity for agonists and does not bind the $ alpha$-toxin. Muscle nAChRs, which also bind $ alpha$-BGT, had been reported to be sensitive to a thymus-derived polypeptide, thymopoietin (TPO). The present work was therefore done to determine whether this agent might also interact with the different nAChR populations in neuronal cells.Initial studies involved thymopentin (TP-5), a 5 amino-acid peptide which may represent the active site of TPO. TP-5 inhibited nicotinic receptor-induced release of catecholamines in bovine adrenal medullary cells in culture, a function mediated through the $ alpha$-BGT-insensitive nAChR. On the other hand, TP-5 did not inhibit either ($ sp3$H) (-)nicotine or ($ sp{125}$I) $ alpha$-BGT binding to rat brain membranes. These results suggested that TP-5 interacted in a non-competitive manner with the $ alpha$-BGT-insensitive neuronal nAChR.
Studies were subsequently done with thymic preparations presumed to be purified TPO ('TPO'), the native polypeptide containing the TP-5 amino acid sequence. In contrast to the effect of TP-5, 'TPO' preparations did not alter nicotinic receptor mediated catecholamine release from neuronal cells in culture. However, 'TPO' preparations selectively decreased ($ sp{125}$I) $ alpha$-BGT binding to brain membranes suggesting an interaction between this polypeptide and $ alpha$-BGT receptors. Quantitative autoradiography revealed that 'TPO' inhibited specific ($ sp{125}$I) $ alpha$-BGT binding uniformly and with similar potency in different brain regions. As $ alpha$-BGT binding sites are highly expressed in the hippocampal formation, primary cultures of fetal rat hippocampal cells were used next to investigate regulation of the $ alpha$-BGT receptor by 'TPO'. 'TPO' caused a dose-dependent and slowly reversible decrease in the density of $ alpha$-BGT receptors. After completion of this work with 'TPO', studies by Quik and coworkers (1993) showed that $ alpha$-Naja toxin (or $ alpha$-cobratoxin) from Naja naja siamensis snake venom was present in the 'TPO' preparations; furthermore, this toxin component appeared to be responsible for the reported effects of 'TPO' on $ alpha$-BGT receptors. Therefore, the above results which had initially been interpreted to occur as a consequence of the interaction of the thymic polypeptide TPO at the nicotinic $ alpha$-BGT site, must now be attributed to the presence of $ alpha$-Naja toxin contaminant in the 'TPO' preparations.
Studies were also undertaken to identify a nicotinic function for $ alpha$-BGT receptors in neuronal cells. Intracellular calcium levels were measured in response to nicotine as recent work using parasympathetic neurons showed that this may represent an $ alpha$-BGT-sensitive response. In contrast to earlier findings, the present work indicates that nAChR-mediated calcium fluxes in cultured chromaffin cells do not reveal an $ alpha$-BGT-sensitive component. These results thus suggest that nicotinic $ alpha$-BGT receptors mediate their response by altering intracellular calcium levels in some but not all neuronal preparations.
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Tandon, Anurag. "Adenosine and acetylcholine synthesis in a sympathetic ganglion." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28539.
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The role of adenosine in modulating synaptic transmission in the cat superior cervical ganglion was investigated in this thesis. The first study showed that perfusion of ganglia with exogenous adenosine increased their acetylcholine (ACh) content. The effect was reduced by blockade of nucleoside transport, as if adenosine's action was mediated at an intracellular site. Isotopic labelling of the extra ACh with radiolabelled choline showed that the additional transmitter was due to increased ACh synthesis, and associated with increased choline transport. After its formation, preganglionic stimulation could release the extra ACh, but not if vesamicol, a vesicular ACh transport inhibitor, was present. Thus, the extra ACh appears to require mobilization from a reserve pool of transmitter.Activity-dependent modulation of synaptic transmission is known to occur in sympathetic ganglia. One such form of adaptive behavior is the increase in ACh content ('rebound ACh') after high frequency preganglionic stimulation. The possibility that adenosine might play a role in the rebound phenomenon was examined in the second study. The accumulation of rebound ACh was sensitive to nucleoside transport inhibitors; dipyridamole reduced rebound ACh if it wzs present only after the stimulation, but not if it was present only during stimulation. After its synthesis, rebound ACh was released by preganglionic stimulation, but not if vesamicol was present, as if the extra transmitter had to be mobilized from a reserve pool. Because the dipyridamole-sensitive step occurred after the conditioning period, it seemed possible that a retrograde messenger triggered the post-stimulation change in ACh synthesis.
Thus, the final series of experiments tested whether a postsynaptic signal could alter presynaptic ACh synthesis. Antidromic stimulation increased ganglionic ACh synthesis, and, consequently, ACh content. Subsequent evoked ACh release was potentiated, as if the additional transmitter was releasable. The antidromic stimulation-induced increase in AZh content was blocked by dipyridamole suggesting that adenosine might be involved.
Overall, the results presented in this thesis are consistent with the notion that adenosine acts as a retrograde messenger after high frequency orthodromic stimulation to induce an increase in presynaptic ACh synthesis.
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Nichols, Philip Paul. "Transcriptional regulation of the human nicotinic acetylcholine receptor." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326016.
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Obosi, Louis A. "Cloning and expression of insect acetylcholine receptor genes." Thesis, Oxford Brookes University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314726.
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Pimlott, Sally L. "Radiosynthesis and evaluation of novel acetylcholine receptor radioligands." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398628.
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Lansdell, Stuart John. "Folding and assembly of neuronal nicotinic acetylcholine receptors." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298829.
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Hammond, Victoria. "α7 nicotinic acetylcholine receptors at the glutamatergic synapse." Thesis, University of Bath, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.633163.
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Nicotinic acetylcholine receptor (nAChR) activation is neuroprotective and nicotine is a cognitive enhancer. Loss of nAChRs, deposition of tau neurofibrillary tangles, cleavage of amyloid precursor protein (APP) and inflammation are well documented in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β- and γ-secretase enzymes generates soluble Aβ peptides, with oligomeric forms of Aβ implicated in both the control of synaptic excitability and dysregulation of synaptic transmission and induction of neuronal death in AD. Aβ production is inhibited by calcium-dependent recruitment of α-secretase, as exemplified by activation of N-methyl-D-aspartate receptors (NMDAR). All neurodegenerative diseases are associated with inflammation, arising from altered homeostasis of the innate immune system, resulting in heightened activation of immune cells and induction of a pro-inflammatory environment. Stimulation of the α7 subtype of nAChR is anti-inflammatory and also enhances cognition and promotes neuronal survival. This work addressed the hypotheses that stimulation of highly calcium-permeable α7nAChR inhibits Aβ production by promoting α-secretase-mediated processing of APP and also modulates inflammatory cellular behaviour of microglia. Thus, this study assessed the role of α7nAChR at glutamatergic synapses, through probing effects on APP processing and phagocytosis in primary cortical neurons and microglia, respectively. Primary cortical neurons expressed functional α7nAChR and glutamate receptors, and through a number of experimental approaches, including immunoblotting and a cleavage reporter assay, results indicated α7nAChR activation with the α7nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596 had no effect on APP and Tau, in contrast to NMDAR activation that significantly modulated these proteins. Data suggest low expression of α7nAChR, coupled with distinct localisation of presynaptic α7nAChR and postsynaptic APP could explain the lack of effect. In addition, primary microglia were highly responsive to lipopolysaccharide and possessed functional α7nAChR that coupled to ERK phosphorylation. Microglial α7nAChR activation promoted neuroprotective phagocytic behaviour, in agreement with the ‘cholinergic anti-inflammatory pathway’. This study supports the hypothesis that α7nAChR are modulators of anti-inflammatory behaviour, thus α7nAChR-selective ligands are viable candidates for the treatment of AD and promoting cognitive enhancement.
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Nickless, Jane Christina. "Cellular immunity to acetylcholine receptor in myasthenia gravis." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767550.
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Nicotinic acetylcholine receptor (AChR) has been purified from Torpedo electric organ, foetal calf muscle, adult human leg muscle, and foetal human skeletal muscle, by extraction in non-ionic detergent followed by affinity purification on immobilised a-toxin. The purified AChR preparations were used to study cellular responses in vitro from patients with myasthenia gravis. In addition, several characterisation studies were carried out on the foetal calf AChR preparation. Purified foetal calf AChR was shown in isoelectric focussing experiments to focus as a single sharp peak at pH 5.2 +/- 0.1, and the receptor sedimented in sucrose density gradients as a single species with a sedimentation coefficient S20w= 9.35 +/- 0.10 S, when indirectly labelled with [125 I]-a-bungarotoxin. SDS-polyacrylamide gel electrophoresis of purified foetal calf AChR consistently showed five major protein bands with (Mr) 40, 44, 47, 52 and 57 K. The 57K protein sub-unit was always the most prominent band. A procedure by which to measure antigen-specific lymphocyte proliferation in vitro was developed and optimised in a system using tetanus toxoid and peripheral blood mononuclear leucocytes (PBL) from a normal donor recently immunised against tetanus. Conditions for the production of human T-cell growth factor (TCGF), or Interleukin 2 (IL-2) were optimised, and used in the tetanus toxoid system to develop a procedure by which antigen-specific reactive T-lymphocytes could be isolated, expanded into long-term lines, and ultimately cloned. Antigen and TCGF were required for the expansion and maintenance of the T-cell lines and clones. The AChR purification procedure was modified several times in order to obtain a workable, non-inhibitory AChR preparation for studies of myasthenia gravis in vitro. Lymphocytes, collected from myasthenic blood samples, were shown to proliferate weakly in response to purified AChR added in vitro, with a mean stimulation index (+/- SEM) of 1.72 +/- 0.12. The proliferative response did not appear to be related to the species of AChR employed, age, sex, or clinical classification of the patient, but higher stimulation indices were obtained from patients in a state of disease exacerbation at the time the sample was taken. T-cell fractionation, or reactive T-cell pre-selection steps, did not enhance the proliferative response of myasthenic lymphocytes to purified AChR in vitro, and attempts to isolate and expand AChR-autoreactive T-lymphocytes using antigen and IL-2 were unsuccessful.
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Patel, Anup B. "Nicotinic acetylcholine receptor ligands from 2,4-methanoproline derivatives." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/29973.
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Since the discovery of the powerful analgesic epibatidine in 1974 from the Ecuadorian poison frog Epipedobates tricolor, there has been global interest in the synthesis of analogue molecules. Epibatidine has the unique 7-azabicyclo[2.2.1]heptane structure with a chloropyridyl ring at the 2-positon. Epibatidine acts at the nicotinic acetylcholine receptor (nAChR) and the aim of this work is to produce target compounds retaining therapeutic potential but with higher nAChR sub-type selectivity and lower toxicity. The only naturally occurring compound to have the 2-azabicyclo[2.1.1]hexane system is the nonproteinogenic amino acid 2,4-methanoproline. This alternative bicyclic framework opens the route to the construction of pioneering epibatidine analogues. The intramolecular [2+2] photocycloaddition method was employed to construct the rigid 2-azabicyclo[2.1.1]hexane skeleton. Successful nucleophilic substitution at a methylene attached to the bridgehead position of the 2-azabicyclo[2.1.1]hexane ring system opened the way to construction of innovative derivatives. These have a wider range of functional groups attached at the 1-positon via a methylene 'spacer' and provide access to epibatidine analogues containing heterocyclic substituents and to further homologation. Mechanistic studies indicate that displacements with loss of a nucleofuge require thermal activation but proceed without the rearrangement initially anticipated in such a strained bicyclic structure. A unique tricyclic carbamate has been isolated; nucleophilic attack on this carbamate leads directly to the isolation of N-deprotected substitution products with concomitant decarboxylation. The analogues produced in this study are currently being pharmacologically tested and the results will determine the course of future synthetic approaches towards original targets.
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Collins, T. "Molecular pharmacological characterisation of neuronal nicotinic acetylcholine receptors." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/624494/.
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Neuronal nicotinic acetylcholine receptors (nAChRs) are excitatory ligand‐gated ion channels that perform important roles throughout the nervous systems of both vertebrate and invertebrate organisms. Impairments to human nAChRs and cholinergic transmission are thought to underlie the pathophysiologies of several neurological and psychological diseases including schizophrenia, Alzheimer’s disease, Parkinson’s disease and certain forms of epilepsy. They are also the receptors that mediate the effects of tobacco smoking and contribute to the physiological and psychological changes associated with nicotine addiction. The aim of this thesis is to further our understanding of neuronal nAChRs from a pharmacological and molecular viewpoint. Research described in this thesis focuses on numerous aspects of neuronal nAChRs; allosteric modulators, insect nAChRs and chaperone proteins. Allosteric modulators of nAChRs are ligands that alter the receptor’s responsiveness to agonists via sites that are separate from the agonist‐binding site (the orthosteric site). Positive allosteric modulators (PAMs) increase the receptor's sensitivity to agonist acetylcholine whereas negative allosteric modulators (NAMs) decrease sensitivity. Using molecular and electrophysiological techniques on the α7 nAChR, studies have been conducted with three PAMs (ivermectin, NS‐1738 and PNU‐120596) and one NAM (methanandamide). Chimeric receptor constructs and site‐directed mutagenesis studies, together with ligand docking simulations, have highlighted the importance of the transmembrane region of the α7 nAChR for modulation by these ligands. The second topic covered by this thesis is insect nAChRs and the molecular chaperone RIC‐3 (resistance to cholinesterase inhibitors); a protein that facilitates folding and assembly of nAChRs. In the past, recombinant expression of insect receptors has proved largely unsuccessful and in some (but not all) circumstances co‐expression with RIC‐3 has alleviated the problems. This research is aimed at investigating the influence of alternatively spliced isoforms of Drosophila melanogaster RIC‐3 on the maturation of a variety Drosophila recombinant nAChR subunits. Lastly, ongoing work is also described on the cloning of insect nAChR subunits from pest species Frankliniella occidentalis (western flower thrip), which has developed resistance to the insecticide spinosad.
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Cai, Yuan. "STRIATAL ACETYLCHOLINE-DOPAMINE INTERACTIONS IN PHYSIOLOGY AND PATHOPHYSIOLOGY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=case16069525687865.
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Ruivo, Leonor M. Teles-Grilo. "Amperometric measurement and optogenetic manipulation of acetylcholine release." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.688215.
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Acetylcholine is one of many neurotransmitters engaged in modulating complex / behaviours. Cholinergic projections from the basal forebrain to the prefrontal cortex and the hippocampus are proposed to have an important role in regulating arousal and enhanced attention states, neuronal network activity, and synaptic plasticity during working memory 'functions. Supporting spatia~ working memory, the hippocampus is responsible for spatial navigation and encoding of new episodic memories, while the prefrontal cortex is engaged in top-down processing and handling of information from multiple sources to create cross-temporal and cross-modal associations that support goal-direct behaviour. With the aim to investigate the relationship between acetylcholine release and behavioural states switches, microelectrochemical biosensors were used to make continuous, sub-second resolution measurements of the spatiotemporal dynamics of acetylcholine release in the medial prefrontal cortex and dorsal hippocampus of mice. Acetylcholine release unfolded at two distinct time scales. Tonic acetylcholine release was coordinated in both brain regions, associated with levels of arousal and was dependent on the sequence of behavioural state transitions. Phasic acetylcholine release occurred during performance of a rewarded spatial working memory task. Phasic release events were detected preferentially in the maze reward delivery points regardless of trial outcome, pointing towards a role for acetylcholine in supporting cue-detection and placereward association. These data represent the first high-temporal resolution profile of acetylcholine release measured continuously across a number of different behavioural states, demonstrating the effectiveness of biosensors as a tool suitable to dissect in detail the role of acetylcholine in modulating brain states and behavioural output in vivo. In order to set up the techniques required to mimic in vivo acetylcholine release in hippocampal slices, optogenetic targeting of the septohippocampal cholinergic system was tested using conditional expression recombinant adeno and adeno-associated viral vectors, ChAT-Cre and channelrhodop$in knock-in mouse lines. Preliminary data on viral vector titration and channelrhodopsin expression time-course showed that transduction efficiency and specificity depended on the viral vector and channelrhodopsin mutant used. Widespread, targeted expression was observed following injections of a serotype-2 adeno-associated viral vector carrying the floxed, mCherry-tagged channelrhodopsin mutant H134R and in ChAT-Ai32 transgenic mice.
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AlSharari, Shakir. "Role of Nicotinic Acetylcholine Receptors in Experimental Colitis." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2895.
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Substantial evidence in the literature shows that tobacco smoking has complex and divergent effects on inflammatory bowel diseases (IBD). It ameliorates ulcerative colitis (UC); whereas it aggravates the risk of Crohn’s disease (CD) and affects the disease course and severity. Studies have shown that nicotine has a positive influence on symptoms of UC. Also, it is demonstrated that nicotinic acetylcholine receptor, especially α7 subunit plays an essential component in the vagus nerve-based cholinergic anti-inflammatory effects. In the present study, we explored the effect of nicotine and α7 nicotinic agonists treatment in the DSS colitis mouse model. We also investigated the effects of cotinine, a major metabolite of nicotine, in the model. Methods: Different groups of C57BL6 mice, as well as α7, α5, and β2 nicotinic receptor knock out mice, and their littermates wild-type nicotinic receptor male adult mice were given DSS solution freely in the drinking water for 7 consecutive days after which tap water was given on the 8th day. We measured a Disease Activity Index (DAI) that includes body weight loss, blood presence in stools, stool consistency, local rectal irritation and length of the colon. The mice were then sacrificed on day 8 to allow examination of the entire colon. Disease severity and colon tissue histology and inflammatory markers including colonic myeloperoxidase (MPO) and colonic tumor necrosis factor-α (TNF-α) were evaluated. Levels of MPO and TNF-α were determined by enzyme-linked immunosorbent assay analysis of the homogenized colon samples. The effect of oral, subcutaneous, mini pump nicotine, and oral cotinine treatments were examined on experimental colitis induced by 2.5% DSS in mice. In addition, we measured the plasma levels of the nicotine and cotinine in our treatment protocols. Results: The DSS 2.5% model of colitis is easily induced in mice. Administration of low doses of oral nicotine (12.5 and 25 μg/ml), but not high doses in DSS-treated mice displayed a significant decrease in disease activity index value, total histological damage scores, as well as colonic level of TNF-α compared to the control group. However, the anti-inflammatory effect of nicotine was not seen with chronic s.c., mini pump nicotine or oral cotinine administration. Differences in plasma levels of nicotine and cotinine do not seem to account for this lack of effect. Moreover, neither nicotine nor cotinine reversed colon length shortening in DSS-treated mice, except with the 0.5 mg/kg s.c. dose of nicotine. There was no change in MPO activity among the groups treated with oral or s.c. nicotine. Cotinine oral administration on its own failed to show a significant effect in the DSS model of colitis. α7 KO mice displayed a significantly increased in DAI value starting from day 4 till day 8, histological damage scores and TNF-α levels of were increased significantly compared to their littermate WT mice. Moreover, pretreatments with PHA-543613 (8 mg/kg), a selective α7 agonist, and choline chloride (40 ug/ml), an α7 nAChR natural agonist, significantly reduced clinical parameters in DSS-treated mice; however, they slightly inhibited the increase in the colonic TNF-α levels compare with vehicle DSS-treated mice. Moreover, PNU-120596 (3 mg/kg), a positive allosteric modulator for α7 nAChRs, significantly reduced DAI value and total histological damage score in DSS-treated mice. Conclusion: Results obtained from this study highlight that dose and route of administration play a critical role in the protective effect of nicotine in the DSS mouse colitis model. Also, these data suggest that α7 nAChR has a protective role in colitis with narrower therapeutic index. Data obtained from this study further understanding of the effect of nicotine in UC and may contribute in the development of new pharmaceutical designs for targeting nAChRs for the treatment of ulcerative colitis.
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Blackhall, Kristina Jane. "The synthesis of agonists to the acetylcholine receptor." Thesis, University of Exeter, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260707.
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Gill, J. K. "Allosteric modulation of alpha 7 nicotinic acetylcholine receptors." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1415907/.
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Nicotinic acetylcholine receptors (nAChRs) are selective ion channels that belong to the Cys-loop superfamily of ligand-gated ion channels. They are expressed in skeletal muscle, where they mediate muscle contraction, and in the peripheral nervous system, where they mediate ganglionic transmission. NAChRs are also widely expressed in the central nervous system, where they regulate the release of acetylcholine and several other important neurotransmitters. Conventional nAChR agonists, such as acetylcholine, act at an extracellular ‘orthosteric’ binding site, located at the interface between two adjacent subunits. Previous studies have identified a series of positive allosteric modulators (PAMs) that lack agonist activity but which are able to potentiate responses to orthosteric agonists such as acetylcholine. It has been shown, for example, that 4-(1-napthyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulphonamide (TQS) acts as a conventional α7 nAChR PAM. In the present study, evidence has been obtained indicating that a closely related compound, 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulphonamide (4BP-TQS) is a potent allosteric agonist in both recombinant and native α7 nAChRs. Studies with nAChRs altered by site-directed mutagenesis support the conclusion that allosteric agonists such as 4BP-TQS act via an intra-subunit transmembrane site. Changes in the chemical structure of allosteric modulators have been found to exert profound effects on their pharmacological properties. For example, replacement of the bromine atom in 4BP-TQS with either a chlorine or iodine atom or methyl group (4CP-TQS, 4IP-TQS and 4MP-TQS) results in compounds with pharmacological properties characteristic of allosteric agonists but which display differences in activation rates, inactivation rates and in levels of desensitisation. Further studies conducted with a series of methyl-substituted compounds have provided evidence that molecules interacting with a transmembrane allosteric site can exert diverse pharmacological properties. These include allosteric activation, potentiation of responses to orthosteric agonists and antagonism of orthosteric agonists. These findings highlight the pharmacological diversity of compounds interacting with nAChRs.
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Haghighi, Ali Pejmun. "Mechanism of inward rectification of neuronal nicotinic acetylcholine receptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0032/NQ64568.pdf.
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Gardner, Kristen. "Novel regulation of cortical acetylcholine release and cognitive behavior." Connect to resource, 2008. http://hdl.handle.net/1811/32226.
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Thesis (Honors)--Ohio State University, 2008.Title from first page of PDF file. Document formatted into pages: contains 46 p.; also includes graphics. Includes bibliographical references (p. 37-46). Available online via Ohio State University's Knowledge Bank.
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Blake, Allan David. "Functional characterization of a cloned Drosophila muscarinic acetylcholine receptor." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.319493.
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Kracun, Sebastian. "Molecular and functional characterisation of nicotinic acetylcholine receptor chimaeras." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444289/.
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Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which exhibit considerable subunit diversity. They have been implicated in processes including synaptic transmission and modulation of neurotransmitter release. They also have a significant role in several pathological disorders as well as nicotine addiction, which makes nAChRs important targets for therapeutic drug discovery. One of the aims of this study was to investigate the influence of the intracellular domain of nAChR subunits upon receptor assembly, targeting and functional properties. A series of subunit chimaeras was constructed, each containing the intracellular loop region, located between transmembrane (TM) domains M3 and M4, from nAChR subunits al-alO or pl-p4 and from the 5-hydroxytryptamine type 3 receptor (5-HT3R) subunits 3 A and 3B. Evidence has been obtained which demonstrates that the large intracellular loop exerts a significant influence upon the levels of both cell-surface and intracellular assembled receptors. Comparisons of functional ion-channel properties revealed a significant influence upon both single-channel conductance and receptor desensitisation. Experiments conducted in polarised epithelial cells demonstrate that the nAChR loop can also influence receptor targeting. In a further study, the influence of the recently identified nAChR molecular chaperone, RIC-3 (resistance to mhibitors of cholinesterase), on receptor maturation was investigated. The influence of subunit domains upon the RIC-3's chaperone activity was investigated by co-expression with subunit chimaeras. Finally, a9/5-HT3A and alO/5-HT3A subunit chimeras were used to investigate the pharmacological properties of a9al0 nAChRs, a receptor subtype expressed in hair cells of the auditory system. Physiologically relevant concentrations of the anti-malarial compounds, quinine, quinidine and chloroquine were shown to act as competitive inhibitors, whereas the NMDA receptor antagonist, neramexane, blocked a9al0 nAChR mediated responses via a non-competitive mechanism.
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Thomas, Rachel. "Investigating allosteric activation of the M1 muscarinic acetylcholine receptor." Thesis, University of Leicester, 2010. http://hdl.handle.net/2381/9298.
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Allosteric ligands of G protein-coupled receptors (GPCRs) bind to sites that are topographically distinct from the orthosteric site. AC-42 and 77-LH-28-1 are functionally selective M1 muscarinic acetylcholine (mACh) receptor allosteric agonists that are able to activate the M1 mACh receptor in the absence of an orthosteric ligand. In the present study, a variety of signalling pathways activated by AC-42 and 77-LH-28-1 have been investigated and compared with those activated by orthosteric agonists in Chinese hamster ovary (CHO) cells recombinantly expressing human M1 mACh receptors. Both orthosteric and allosteric agonists are able to activate Gαq/11-dependent signalling as demonstrated by concentration-dependent increases in [35S]-GTPγS binding to Gαq/11 subunits, [³H]-inositol phosphate accumulation and Ca²+ mobilisation. Both AC-42 and 77-LH-28-1 are also able to activate extracellular signal-regulated kinase 1/2 and cyclic AMP response-element binding protein (CREB). However, while all agonists enhance forskolin-stimulated cyclic AMP accumulation, only orthosteric agonists cause significant increases in [35S]-GTPγS binding to Gαi-proteins, suggesting that subtle differences may exist in the receptor conformations stabilised by orthosteric versus allosteric ligands. The effects of orthosteric and allosteric agonists on the regulation of the M1 mACh receptor expressed in CHO cells revealed that in contrast to orthosteric agonists, which cause significant internalisation and down-regulation, prolonged exposure to AC-42 does not significantly alter either cell-surface or total cellular M1 mACh receptor expression. 77-LH-28-1 does cause receptor internalisation, but not down-regulation. The apparent inability of AC-42 to cause M1 mACh receptor desensitisation is supported by the observation that arecoline was still able to stimulate a similar phosphoinositide hydrolysis response in CHO-hM1 cells incubated for 24 h with AC-42. These data indicate that AC -42 binding causes functional signalling in the absence of receptor regulatory mechanisms. These distinct pharmacological properties of allosteric agonists may provide therapeutic advantages additional to receptor subtype selectivity of action.
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Van, Rensburg Ruan. "Upregulation of neuronal α7 nicotinic acetylcholine receptors and preconditioning." Thesis, Durham University, 2007. http://etheses.dur.ac.uk/2452/.
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The upregulation of alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) are putatively reported to play a role in in vivo cortical spreading depression-elicited neuroprotection. In this study, a reliable in vitro spreading depression model was created for studying this phenomenon. In contradiction to previous studies, it was, however, shown that functional α 7 nAChRs are down-regulated upon chronic depolarisation with KCl, although the activity of this receptor subtype remained essential for the preconditioning mechanism. Evidence was provided for a differential mechanism underlying protection against NMDA-mediated and hypotonic-shock induced cell loss. Non-pharmacological upregulation of the α 7 nAChRs in pure neuronal cortical cultures by means of a recombinant adenovirus led to the increased cell death subsequent to an excitotoxic glutamate insult. In addition, in order to study the relationship between α7 nAChRs and its function-dependent regulator Ric3, a novel anti-Ric3 antibody and a recombinant adenovirus expressing the ric3 gene were created. Ric3 was found to be expressed in many important brain structures, including hippocampus, perhinal cortex, thalamic and hypothalamic paraventricular nucleus, structures implicated in both cognitive and emotional behaviours. Interestingly, Ric3 was also expressed in the choroid plexus, a non-neuronal cell type not known to α 7 nAChRs, indicating additional roles for this protein. The recombinant constructs expressing ric3, α 7 nAChRs and dual ric3/ α 7 nAChRs were all validated in vitro.
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Chadha, Preetpal. "Endothelial nicotine acetylcholine receptors : a role in vascular function?" Thesis, University College London (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498105.
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Charriez, Christina Margaret. "ALPHA7 NICOTINIC ACETYLCHOLINE RECEPTOR REGULATION IN EXPERIMENTAL NEURODEGENERATIVE DISEASE." UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/19.
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The α7 nicotinic acetylcholine receptor (nAChR) is involved in learning and memory, synaptic plasticity, neuroprotection, inflammation, and presynaptic regulation of neurotransmitter release. Alzheimer’s disease (AD), a neurodegenerative disease characterized by diminished cognitive abilities, memory loss, and neuropsychiatric disturbances, is associated with a loss of nAChRs. Similarly, traumatic brain injury (TBI) may result in long term neurobehavioral changes exemplified by cognitive dysfunction. Deficits in α7 nAChR expression have previously been shown in experimental TBI and may be related to cognitive impairment experienced in patients following TBI.
The purpose of this dissertation was to investigate changes in α7 nAChR expression in models of neurodegeneration and determine if allosteric modulation of the nAChR facilitates functional recovery following experimental TBI through changes in nAChRs. Experimental models employed include a transgenic mouse model of AD that overexpresses the amyloid precursor protein (APPswe mice) and the controlled cortical impact injury model of TBI in rats. Quantitative receptor autoradiography using α-[125I]-bungarotoxin and [125I]-epibatidine and in situ hybridization were used to investigate changes in nAChR density and mRNA expression, respectively.
In the first study, the effects of aging and β-amyloid on α7 nAChR expression were evaluated in APPswe mice. Hippocampal α7 nAChR density was significantly upregulated in APPswe mice compared to wild-type mice. It is postulated that elevated Aβ levels bind to the α7 nAChR resulting in upregulation. In a second study, galantamine, a medication used in the treatment of AD, was administered subchronically following experimental TBI to determine if treatment could facilitate cognitive recovery and affect nAChR expression. Interestingly, the results indicate TBI interferes with agonist mediated upregulation of nAChRs, and galantamine did not improve function in a behavioral task of learning a memory. In a third study, the regulation of TBI related deficits in α7 nAChRs was examined 48 hours following injury. α7 nAChR deficits occurred with a reduction in α7 mRNA in several hippocampal regions and non-α7 nAChR deficits occurred with a reduction in α4 mRNA in the metathalamus. The results of these studies suggest AD and TBI may involve complex but parallel processes contributing to the regulation of α7 nAChRs.
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Bere, Harding Court Edmund de la. "Functional characterisation of expressed ɑ9ɑ10 nicotinic acetylcholine receptor channels." Thesis, University of Bristol, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.652042.
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The primary aim of this project was to investigate receptor and ion channel coupling in a heterologous expression system, the GH4C1 cell line. The primary focus was the interaction between the a9alO nicotinic acetylcholine receptor (nAChR) and the small conductance calcium (Ca2+) -sensitive potassium (K+) channel, subtype 2 (SK2). In both inner hair cells (rnCs) and outer hair' cells (OHCs), the a9alO nAChR and the SK2 channel are spatially and functionally associated, or 'coupled'. Both the a9alO nAChR and SK2 were transiently expressed in vitro and functionally evaluated. The channels were then coexpressed and the interaction between the two was examined electrophysiologically. Channel blockers, receptor antagonists and an intracellular Ca2+ chelator were used, in conjunction with electrophysiological methods. No evidence of the interaction between the a9alO nAChR and the SK2 channel was found. Prior to the investigation of the coupling interaction, the endogenous currents of the GH4C1 cells were also assessed. Other experiments were aimed at recapitulating the interaction of an L-type Ca2+ channel (LTCC) and the SK2 channel in vitro, which also occurs in neonatal IHCs. These channels were not found to associate, in the cell line. Additionally, the direct effect of ryanodine upon the SK2 channel was assessed in Human embryonic kidney (HEK) 293 cells, and found to have no effect.
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Kirwan, Michael Joseph. "Molecular cloning and characterisation of insect nicotinic acetylcholine receptors." Thesis, University College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408548.
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Purohit, Prasad G. "Molecular determinants of pharmacological distinctions among nicotinic acetylcholine receptors." Thesis, University of Strathclyde, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424352.
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Cross, Kathryn Mary Louise. "Studies on nicotinic acetylcholine receptors expressed in surrogate cells." Thesis, University of Southampton, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295914.
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Stephens, Mark William. "Pharmacological characterisation of the α4 neuronal nicotinic acetylcholine receptor." Thesis, University of Bath, 1993. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760649.
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Macallan, David Robert Edward. "Studies on the nicotinic acetylcholine receptor of the locust." Thesis, University of Bath, 1988. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760577.
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