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1
BAVO, FRANCESCO. "SUBTYPE-SELECTIVE NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR AGONISTS AND ANTAGONISTS." Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/607332.
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This PhD thesis focuses on two specific targets, belonging to the same receptor class of Nicotinic Acetylcholine Receptors (nAChRs): the α4β2 subtype and the α7 subtype. This elaborate is divided in two parts. The aim of the first part is the design and synthesis of α4β2 selective partial agonists as potential smoking-cessation agents. The aim of the second part is the design and synthesis of α7 antagonists with mitocan properties as antitumoral agents.
Part 1.
In the first project, a series of 3-nitrophenyl ethers and 3-hydroxyphenyl ethers of (S)-N-methylprolinol bearing bulky and lipophilic substituents at the C5 were designed, synthesized and assayed as putative selective α4β2 ligands. Two of them, 5-substituted with a 6-hydroxy-1-hexynyl, had high α4β2 affinity and increased α4β2/α3β4 selectivity when compared with the correspondent unsubstituted parent compounds.
In the second project, each -CH= of the unselective antagonist (S,R)-N-methyl-2-pyrrolidinyl-1,4-benzodioxane and of its epimer at the benzodioxane stereocenter, was replaced by a nitrogen. The resulting four diastereomeric pairs of pyrrolidinyl-pyridodioxanes, also designed as the product of rigidification of the flexible scaffolds of pyridyl ethers of N-methyl prolinol, were studied for their nicotinic affinity at the α4β2 and α3β4. The isosteric -CH= to N substitution was detrimental for all the compounds, with the only exception of N-Methyl-pyrrolidinyl 5-pyridodioxane, with the nitrogen at position 5. Indeed, this ligand had similar affinity to its benzodioxane parent compound, but it had high α4β2/α3β4 selectivity and it was shown to be a selective partial agonist.
In the third project, the unselective antagonist (S,R)-N-methyl-2-pyrrolidinyl-1,4-benzodioxane and of its epimer at the benzodioxane stereocenter were substituted at position 5 of the benzodioxane moiety, to explore the possibility of introducing selectivity and/or partial agonist as previously done with -CH= to N replacement. Among the synthesized compounds, (S,S)-N-Methyl-pyrrolidinyl-5-amino-benzodioxane had slightly improved affinity at the α4β2 affinity and highly enhanced α4β2/α3β4 selectivity than the unsubstituted parent compound, and it was shown to be a very potent partial agonist.
In the fourth project, we applied computational techniques to support the interpretation of the biological results regarding N-Methyl-pyrrolidinyl 5-substituted benzodioxanes and pyridodioxanes. From these findings, we suggested that partial agonism and α4β2/α3β4 selectivity could be achieved when the benzodioxane scaffold is appropriately substituted with an HBA/HBD system, that can displace a water molecule from a small and hydrophilic subpocket of the binding site.
Part 2.
Adenocarcinoma and glioblastoma cell lines express α7 and α9-α10 nAChRs, whose activation promotes tumor cells growth. On these cells, the triethylammoniumethyl ether of 4-stilbenol MG624, a known selective antagonist of α7 and α9-α10 nAChRs, has antiproliferative activity. The structural analogy of MG624 with the mitocan RDM-
4’BTPI, triphenylphosphoniumbutyl ether of pterostilbene, suggested us that molecular hybridization among their three substructures might result in novel antitumour agents with higher potency and selectivity. We found that replacement of ethylene with butylene in the triethylammonium derivatives results in more potent and selective toxicity towards adenocarcinoma and glioblastoma cells, which was paralleled by increased α7 and α9-α10 nAChR antagonism and improved ability of reducing mitochondrial ATP production. Further elongation to octylene (26) provided a compound with 40-fold and 10-fold increased antiglioblastoma and antiadenocarcinoma activity respectively, when compared to MG624. Elongation of the alkylene linker was greatly advantageous also for the triphenylphosphonium derivatives. RDM-
4’BTPI did not acquire, as expectable, antinicotinic activity by hybridization with MG624 stilbene scaffold, but it was surpassed in glioblastoma cell viability reduction by its stilbene analogues with > 4C alkylene linker. In particular, the analogue with decylene between stilbenoxyl and triphenylphosphonium head (24) was ten-
fold and two-fold more potent than RDM-4’BTPI in reducing glioblastoma cell viability and in increasing ROS production respectively. Overall, the ammonium compound
26 reached antiproliferative activities at glioblastoma and adenocarcinoma cells in the same range of the phosphonium 24, but showed good selectivity against neuroblastoma and healthy mouse astrocytes.
2
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).
3
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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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.
5
Larsen, James D. "Nicotinic Acetylcholine Receptor Dependent Effects of Nicotine on HEK293T and HBO Cells." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5701.
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T2R receptors are the classical bitter taste receptors which detect and transduce bitter taste in a subset of taste receptor cells (TRCs). The TRPM5-dependent T2Rs are G-protein coupled receptors (GPCRs) and are linked to G protein, gustducin to initiate an intracellular signaling cascade for the transduction of bitter tastants. Nicotine is bitter. However, at present the transduction mechanisms for the detection of nicotine in are poorly understood. Previous studies from our laboratory using TRPM5 knockout (KO) mice demonstrated that the T2R pathway is insufficient in explaining the taste perception of nicotine. TRPM5 KO mice elicited chorda tympani (CT) taste nerve responses to nicotine, albeit significantly smaller than the wild type (WT) mice and still responded to nicotine as an aversive stimulus. Following addition of mecamylamine (Mec), a non-specific blocker of neuronal nicotinic acetylcholine receptors (nAChRs), CT responses to nicotine were partially inhibited in both WT and TRPM5 KO mice. Mec also decreases the aversive response to nicotine in both WT and TRPM5 KO mice. These studies led to the hypothesis that both a TRPM5-independent and TRPM5-dependent pathways are responsible for the detection and transduction of the bitter taste of nicotine in TRCs. The TRPM5-independent pathway most likely utilizes the nAChRs expressed in TRCs and function as bitter taste receptors for nicotine. We have subsequently demonstrated the expression of nAChRs in a subset of TRPM5-positive TRCs. However, this mechanism is not well understood in other cell types, particularly undifferentiated epithelial cells, such as HEK293T cells. The specific aims of this project were: (i) To identify which components of T2R-dependent taste reception as well as components of nAChRs are expressed in HEK293T cells; (ii) To determine if HEK293T cells co-express these components; (iii) To identify if exposure to nicotine modulates the expression of T2R and nAChR dependent components in HEK293T cells; (iv) To determine if TRCs express functional nAChR ion channels; and (v) To determine if nAChRs are involved in the release of neuropeptides, such as brain-derived neurotrophic factor (BDNF) in HEK293T cells. The data obtained in HEK293T cells was compared with parallel studies on adult cultured human fungiform taste cells (HBO) done independently by Dr. Jie Qian, a postdoctoral fellow in Dr. Vijay Lyall’s lab. The results of combined studies on HBO and HEK293T cells indicates that TRPM5-positive cells also co-express ionotropic nAChRs, comprising a and β subunits. The nAChRs are capable of forming ion pores and when stimulated by nicotine and create a parallel TRPM5-independent pathway for the detection of nicotine. Using molecular and immunocytochemical techniques, our results demonstrate that mRNAs and proteins for bitter taste receptors and downstream intracellular signaling components as well as subunits necessary for the formation of nAChRs are expressed in HBO and HEK293T cells. Results demonstrated that TRPM5-positive HEK293T cells co-expressed nAChR subunits throughout the entire population. Nicotine increased the influx of Ca2+ in a dose dependent manner, which was somewhat reduced by the addition of TRPM5 blocker, triphenylphosphine oxide (TPPO). Both mRNA and protein expression were altered in a biphasic pattern with a maximum increased observed at 0.5 µM nicotine with a decrease in expression at higher concentrations. The synthesis of neurotrophic factor BDNF, required for maturation of taste bud cells and their innervating nerves, increased in HEK293T cells exposed to nicotine, however, nicotine did not trigger the release of BDNF. These results were then compared and contrasted with HBO cells to better understand the comparative effects of nicotine on both undifferentiated and differentiated cells. The data on HBO cells is presented in the Appendix.
6
Brown, Jack. "α7 Nicotinic acetylcholine receptor-mediated calcium signalling in neuronal cells." Thesis, University of Bath, 2014. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636524.
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α7 nicotinic acetylcholine receptors (nAChR) are highly permeable to Ca2+ and are clinical targets for Alzheimer’s disease and schizophrenia. The aim of this work was to examine α7 nAChR-mediated Ca2+ signalling in neuronal cells using three different methods, and to evaluate the effects of the desensitizing agonist and prototypical smoking-cessation drug sazetidine-A on α7 nAChRs. Initial studies used 96-well plate assays with SH-SY5Y cells to characterize responses evoked by the α7 nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596. This was complemented by live-imaging of cortical cultures, where the compounds evoked robust Ca2+ responses from 12 % of cells. Co- application with Cd2+, ryanodine and xestospongin-C significantly inhibited these responses, suggesting the involvement of voltage-gated Ca2+ channels and Ca2+- induced Ca2+-release. CNQX and MK801 also significantly inhibited α7 nAChR mediated Ca2+ elevations, indicating a role for glutamate release. A high-content screening assay was developed to further examine these phenomena. Exploratory experiments using KCl, AMPA and NMDA validated a protocol that could be used to image Ca2+ elevations in large cell populations. Inconsistent responses to PNU-120596 and PNU2-282987 were also observed, reflecting the scarcity of α7 nAChRs in cortical cultures and the need for assay optimization. Combination with immunofluorescent labelling revealed α7 nAChR mediated Ca2+ elevations in a subpopulation of astrocytes and neurons, some of which were GABAergic. PNU-120596 potentiated the effects of sazetidine-A in SH-SY5Y cells (EC50 0.4 μM) eliciting responses in 14 % of cells in cortical cultures in a methyllycaconitine- sensitive manner, consistent with α7 nAChR activation. Pre-incubation with sazetidine-A concentration-dependently attenuated subsequent α7 nAChR-mediated responses in SH-SY5Y cells (IC50 476 nM) and cortical cultures, suggesting that α7 nAChRs could play a role in the behavioural effects of sazetidine-A. These comparative experiments enhance our understanding of α7 nAChR signalling and provide a new method to study them further.
7
Covernton, Patrick John O'Neill. "Functional aspects of neuronal nicotinic receptor diversity." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321967.
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Zirger, Jeffrey M. "Cloning, Expression and Functional Analysis of the Zebrafish Neuronal Nicotinic Acetylcholine Receptor." The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1052847984.
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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.
10
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.
12
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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Eastham, Helen Margaret. "Characterisation of the nicotinic acetylcholine receptor from Manduca sexta." Thesis, University of Bath, 1996. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321845.
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Anderson, Shawn. "BETA 2 NICOTINIC ACETYLCHOLINE RECEPTOR CONTRIBUTIONS TO ANXIETY-LIKE BEHAVIOR." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/563.
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Nicotine is a major psychoactive ingredient in tobacco that is thought to promote smoking behavior via nicotinic acetylcholine receptors (nAChRs) in the brain. Given reports that people smoke to relieve anxiety and that anxiety precipitates relapse, the overarching goal of this dissertation research is to assess beta 2 subunit containing nAChR (beta2*nAChR) contributions to anxiety-like behavior. Nicotine’s activity at beta2*nAChRs is concentration-dependent, with high concentrations facilitating activation followed by rapid desensitization and low concentrations preferentially desensitizing beta2*nAChRs; hence, activation or inhibition of beta2*nAChRs may support smoking behavior. Rodent studies reveal that nicotine affects anxiety-like behavior dose-dependently: low doses promote anxiolysis- and high doses support anxiogenic-like behavior. These pharmacological and genetic studies in mice test the hypothesis that nicotine administration promotes anxiolysis via inactivation of beta2*nAChRs and begin to identify which subunits, namely alpha 4 and alpha 6, work in concert with beta 2 to affect anxiety-like behavior. Low dose nicotine and inhibition of beta2*nAChRs supported anxiolysis-like behavior in a number of tasks with predictive validity for anxiolysis efficacy. These studies further suggest that activation of alpha6beta2*nAChRs is sufficient to produce anxiogenic-like behavior and that inhibition of alpha4beta2*nAChRs supports anxiolysis-like behavior. A secondary goal of these studies is to assess if beta2*nAChRs affect anxiety-like behavior during aging. Dysregulation of cholinergic tone can increase anxiety in the elderly, but little is known regarding beta2*nAChR contributions to anxiety in this population or where in the brain this may take place. These studies show that alpha4beta2*nAChR expression differentially affects anxiety-like behavior in adult and aged mice. With a focus on the lateral septum, a GABA-ergic limbic nucleus thought to regulate anxiety-like responses to external stimuli, a third goal of these studies is to elucidate the neuroanatomical and intracellular underpinnings of anxiety-like behavior that are affected by beta2*nAChR inhibition and expression. Previous studies demonstrate that exposure to stressors reduces phosphorylation of extracellular regulated kinase (ERK) in the lateral septum. In these studies, levels of pERK in the lateral septum were inversely associated with alpha4beta2*nAChR expression as well as anxiogenic-like behavior. In sum, these preclinical studies suggest that inhibition alpha4beta2*nAChRs may support cessation in those who smoke to relieve anxiety.
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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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Young, Jared W. "Nicotine induced improvements in cognition : a possible role for the α7 nicotinic acetylcholine receptor." Thesis, University of Edinburgh, 2005. http://hdl.handle.net/1842/27731.
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Assessment of sustained attention in rodents can be performed using the 5-choice serial reaction-time (5-CSR) task; analogous to the continuous performance test used in man. A 5-CSR protocol was established which allowed the demonstration of nicotine-induced improvements in sustained attention in mice. In this task α7 nAChR knockout (KO) mice exhibited impaired acquisition and performance, providing additional evidence that this receptor may be a valid therapeutic target for cognitive enhancement. In order to investigate the role of nAChR manipulation on working memory, the odour span task, a test of olfactory working memory capacity, was established in mice. Nicotine administration did not improve performance of C57B1/6J mice probably as a consequence of ceiling effects. Transgenic mice over-expressing human caspase-3 (hc-3) displayed a robust impairment in the task that was attenuated by nicotine administration. Moreover α7 nAChR KO mice exhibited impaired acquisition and performance in the task but in a different pattern to that of the hc-3 mice. This pattern may reflect an impaired ability to attend to the task as opposed to a working memory deficit alone. These demonstrations provide further support for a role of the α7 nAChR in cognition. Tg2576 mice represent the best well characterised transgenic model of AD, however there remains a dearth of information on their attentional and olfactory capabilities. The mice exhibited a deficit in sustained attention in the 5-CSR task, as well as an age-related impairment in the odour span task. In conclusion the development of the 5-CSR task for mice was used to identify a nicotine-induced improvement in normal mice and impaired performance in α7 KO and Tg2576 mice. In summary these data provide some evidence for a role of the α7 nAChR in nicotine-induced improvement in cognition, and with the tasks developed provide new tools for the assessment of putative cognitive enhancing compounds.
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Barrantes, Georgina Elida. "Nicotinic acetylcholine receptor subtypes in primary cultures of hippocampal neurons." Thesis, University of Bath, 1994. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386845.
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Williamson, Sally. "Nicotinic acetylcholine receptors from the parasitic nematode Ascaris suum." Thesis, University of Bath, 2008. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501371.
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Nematodes of the genus Ascaris are large gastrointestinal parasites. Ascaris lumbricoides infects ~1 billion people globally; causing malnutrition and general morbidity, and can block the gut or bile duct causing fatal complications. Ascaris suum is a parasite of pigs; in addition to its veterinary significance, it can occasionally be zoonotic, and is a good model of the human parasite. One of the main classes of drugs used to treat parasitic nematode infections are the cholinergic anthelmintics, such as levamisole and pyrantel, which act as agonists of nicotinic acetylcholine receptors at the nematode neuromuscular junction.
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Ayers, Joshua Thomas Longen. "SYNTHESIS AND EVALUATION OF PYRIDINIUM DERIVATIVES AS CENTRAL NERVOUS SYSTEM NICOTINIC ACETYLCHOLINE RECEPTOR LIGANDS." UKnowledge, 2006. http://uknowledge.uky.edu/gradschool_diss/414.
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This project utilized synthesis and in vitro assays to generate antagonist SARs at various nAChR subtypes. Alkylation of the pyridino nitrogen of the nicotine molecule afforded subtype specific antagonists at a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Using this data, a series of mono-azaaromatic quaternary salts were produced and evaluated in binding and functional assays for a42* and a7* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. Additionally, bis-azaaromatic quaternary salts were synthesized and evaluated in the same assays. Two potent lead compounds were identified. N-n-dodecylnicotinium iodide (NDDNI) was found to be very potent at both a42* nAChR subtypes and nAChR subtypes that mediate nicotine-evoked dopamine release. And the most promising candidate was N-N-bisdodecylpicolinium dibromide (bDDPiB), which was selective for the nAChR subtypes that mediate nicotine-evoked dopamine release (IC50 = 9 nM). Additionally, using the data from the SARs, predictive computer models were generated to assist in future compound assessment without in vitro assays. Three self-organizing map (SOMs) models were generated from three different sets of compounds. The groups consisted of the mono-substituted compounds, the bissubstituted compounds, and both sets combined. The models were able to successfully "bin" the test set of compounds after developing a model from a similar set of training compounds. Additionally, using genetic functional activity (GFA) algorithms an evolutionary approach to generating predictive model equations was applied to the compounds. Three separate equations were generated in order to form a predictive method for evaluating affinities at the a4b2* receptor subtype. In addition to the modeling and SAR work of the quaternary ammonium compounds, novel synthetic methods were also employed to develop enantiomerically pure nicotine analogs. Efficient enantioselective syntheses of (S)- and R-(+)-nornicotine, (S)-and R-(+)-anabasine, and (S)-and R-(+)-anatabine have been developed, affording isomers in high enantiomeric excess.
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Hibbs, Ryan E. "Conformational dynamics of the acetylcholine binding protein, a Nicotinic receptor surrogate." Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2006. http://wwwlib.umi.com/cr/ucsd/fullcit?p3237010.
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Thesis (Ph. D.)--University of California, San Diego, 2006.Title from first page of PDF file (viewed December 8, 2006). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Hill, Danny G. "Transmembrane domain orientation and neurotransmitter binding in the nicotinic acetylcholine receptor." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26922.
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The orientation of the hydrogen-deuterium exchange resistant "core" of the of the intact nicotinic acetylcholine receptor from Torpedo was studied using linear dichroism attenuated total reflectance infrared spectroscopy both in the presence and absence of the agonist Carb. Our results show that the hydrogen exchange resistant alpha-helical peptide hydrogens in the nAChR are preferentially oriented parallel to the bilayer normal, therefore, providing evidence for a predominantly alpha-helical transmembrane domain, and that there are no detectable net changes in orientation upon agonist-induced desensitization. We also examined the linear dichroism of lipid bands in spectra recorded from membranes both with and without the nAChR in order to accurately assess the effects of membrane film mosaic spread on the interpretation of the linear dichroism data. Our data also show that the mosaic spread of the reconstituted membrane films is greater than the mosaic spread observed with pure lipid films. Our work illustrates the importance of mosaic spread characterization when interpreting experimental order parameters in terms of molecular structure orientation. (Abstract shortened by UMI.)
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Odeh, Rula S. "Thymopoietin and MyoD : their effects on the muscle nicotinic acetylcholine receptor." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=68233.
Full textAbstract:
The present study was done to determine whether the muscle nicotinic acetylcholine receptor (nAChR) function and expression could be regulated by two different factors: (a) thymopoietin (TPO)$ sp *$, a nicotinic antagonist and (b) MyoD, a myogenic transcription factor.Exposure of rat neonatal muscle cells in culture to TPO on a long-term (days) and short-term (minutes) basis resulted in the inhibition of $ sp{125}$I-$ alpha$-bungarotoxin (BGT) binding. Short-term pretreatment with TPO also led to a decrease in carbachol-stimulated $ sp{22}$Na uptake; however, long-term exposure resulted in an enhanced carbachol-stimulated uptake. Chronic treatment also resulted in greater muscle cell morphological development. These results suggest that TPO regulates the nAChR and exerts trophic effects on myotube morphology.
As another approach to study factors that affect nAChR expression, non-muscle cells were transfected with MyoD cDNA. After transfection, saturable, high affinity $ sp{125}$I-$ alpha$-BGT binding was readily detectable, as was carbachol-stimulated $ sp{22}$Na uptake. Both these parameters developed in parallel over time and were inhibited by nicotinic antagonists. These results suggest that the transfection of a non-muscle cell line with MyoD cDNA results in the expression, at the cell surface, of a functional muscle-type nAChR.
This work shows that the nAChR function and expression can be regulated through (a) the chronic interaction of TPO at the nAChR at the cell surface and (b) the action of MyoD at the gene level. ftn$ sp *$ As stated in the addendum to this thesis, recent work by Quik et al. 1993a has shown that the preparation presumed to be TPO, contained $ alpha$-cobratoxin; the effects observed in the present thesis must therefore now be attributed to the presence of $ alpha$-cobratoxin contaminant.
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Wood, Susan C. "Molecular mechanisms of anaesthetic action in nicotinic acetylcholine receptor-rich membranes." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302901.
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Mongan, Nigel Patrick Edward. "The nicotinic acetylcholine receptor gene family of the nematode Caenorhabditis elegans." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624516.
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Kennett, Alexandra. "Nicotinic acetylcholine receptor modulation of noradrenaline release in the rodent brain." Thesis, University of Bath, 2011. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557794.
Full textAbstract:
Cognitive function in the brain is controlled by neurotransmitters whose release is tightly controlled. When normal levels are perturbed deficits in function can be observed both in humans and in animal models. The cholinergic system, acting via muscarinic or nicotinic receptors, modulates neurotransmitter release. The aim of this thesis was to investigate the identity of the nicotinic acetylcholine receptor (nAChR) subtypes involved in modulating noradrenaline (NA) release, in rodent frontal cortex (FC) and hippocampus (HC). Comparisons were made both in vitro and in vivo using pharmacological tools. In vitro, acute application of nicotinic agonists evoked release of previously loaded [3H]-NA from prisms of rat FC and HC. There was a 2000-fold more potent response to β2* selective nAChR agonist 5-iodo-A85380 in FC than HC. A greater response to choline in HC than FC, combined with a lack of response to selective α7 ligands supports α3β4* nAChRs as the main mediator of nicotinic stimulated NA release in vitro in HC. A proportion of the release in each region was mediated via a potentially excitatory action of GABA. The profile of responses was unchanged after the acute or chronic administration of nicotine in vivo. In vivo microdialysis experiments were designed to test whether the nAChR subtype differences in vitro were representative of differences in vivo. 5-iodo-A85380 administered by reverse dialysis increased NA levels to a greater extent in FC than HC, supporting differences in the nAChR composition involved in NA regulation between these two regions. Targeted stimulation of these different nAChR subtypes could allow exploitation of this disparity to improve function with novel compounds such as those described in Chapter 2. Overall the studies described in this thesis show that there are differences in the subtype of nAChRs involved in NA release from terminal fields of FC and HC both in vitro and in vivo.
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Stafford, Alexandra M. "alpha6 beta2 subunit containing nicotinic acetylcholine receptor contributions to abuse-related effects of nicotine and alcohol." VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4778.
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Pharmacotherapies for tobacco and alcohol cessation are only modestly successful, so it is important to better understand mechanisms underlying their use and abuse. The overarching goal of this research is to assess a6b2 subunit containing nicotinic acetylcholine receptor (a6b2*nAChR; *denotes possible assembly with other subunits) contributions to abuse-related effects of nicotine and alcohol. In the absence of a6b2*nAChR-selective agonists, a6b2*nAChR gain-of-function (a6L9’S) mice provide a tool for selective activation of a6b2*nAChRs. Using the a6L9’S mice together with nicotine doses sub-threshold for stimulation of native nAChRs, these studies tested the hypothesis that activation of a6b2*nAChRs is sufficient to promote neurochemical and behavioral effects relevant to nicotine addiction. Intracranial infusions of an a6b2*nAChR-selective antagonist further tested the neuroanatomical locus of a6b2*nAChR contributions to mesolimbic dopamine (DA) release and nicotine reward behavior. Our in vivo microdialysis and nicotine conditioned place preference (CPP) studies reveal that stimulation of a6b2*nAChRs on ventral tegmental area (VTA) DA neurons, as well as on DA terminals in the nucleus accumbens (NAc) shell support nicotine reward. VTA a6b2*nAChR stimulation is required for elevated basal NAc DA levels in a6L9’S mice, who also show elevated nicotine CPP. These studies also showed elevated anxiety-like behavior in a6L9’S mice, but no change in a6 subunit null mutant (a6KO) mice to suggest that elevated cholinergic tone at a6b2*nAChRs promotes anxiety-like behavior. To better define the molecular make-up of a6b2*nAChRs supporting nicotine reward and anxiety-like behavior, these studies crossed a6L9’S to a4 subunit knockout mice to differentiate (non-a4)a6b2* and a4a6b2*nAChR contributions. (non-a4)a6b2*nAChRs appear to promote nicotine reward behavior, while the a6b2*nAChR subtype that regulates anxiety-like behavior depends on the anxiety assay. Finally, these studies developed a mouse model of oral operant ethanol (EtOH) self-administration and assessed EtOH reinforcement in a6 heterozygous (a6HET) and a6KO mice to characterize the role of a6b2*nAChRs in EtOH reinforcement. EtOH self-administration was similar to wild type mice in a6KO mice, but not a6HET mice, suggesting that expression of a6b2*nAChRs modulates EtOH reinforcement. Together, these preclinical studies implicate a6b2*nAChRs in various abuse-related effects of nicotine and alcohol, identifying this receptor as a potential therapeutic target for treatment of dependence.
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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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Moroni, Mirko. "Characterization of alternate forms of the α4�2 nicotinic acetylcholine receptor." Thesis, Oxford Brookes University, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.491310.
Full textAbstract:
Ligand-gated ion channels (LOIC) mediate fast synaptic transmission in the central nervous system and at the neuro-muscular junction. In addition, LOIC can also influence neuronal signalling by influencing the release of neurotransmitters from presynaptic ternlinals. Neuronal nicotinic acetylcholine receptors (nAChRs) are wellknown examples of LOIC that modulate neurotransmitter release. These receptors are located mostly presynaptically in neurones, and due to their high calcium pernleability and fast activation upon ligand binding, they represent a key component of the physiological mechanisms that regulate neurotransmitter release and therefore neuronal signalling. This thesis is concerned with the properties of the a4f32 nAChR, the most abundant nAChR in the brain. The a4f32 nAChR is involved in a variety of physiological tasks, such as nociception, memory and vision, but it is also associated with pathological states such as Alzheimer's disease, Parkinson's disease, schizophrenia and autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Furthermore, due to its major contribution to the central effects of nicotine, the a4f32 nAChR is one of the most studied brain nAChR. The a4f32 nAChR is made up of five subunits, but its stoichiometry remained uncertain until recent insights. In this study, by using site-directed mutagenesis and the voltage-clamp technique, the stoichiometric arrangements of this receptor have been determined by heterologous expression in Xenopus oocytes. It can be concluded that two different arrangements in Xenopus oocytes two homogenous populations are produced: (a4))(f32h and (a4)2(f32)). The pharmacology of the two receptor subtypes has been characterised and subtype-selective drugs were used to investigate the effect of chronic nicotine exposure on both stoichiometric arrangements. The data presented here support the idea of nicotine acting as a chaperone in the endoplasmic reticulum, driving the receptor assembly of the a4 and f32 subunits towards the (a4)2(f32h stoichiometry. This thesis also examined the sensitivity of both a4f32 receptor stoichiometries to modulation by 2n2+. 2n2+ is the second most abundant ion present in the brain and its physiological relevance in modulating fast synaptic transmission has been widely documented. Here it is shown that the stoichiometry of the a4J32 receptor determines how the receptor responds to 2n2+: 2n2+ only inhibits (a4h(f32)) receptors, whilst it potentiates or inhibits, depending upon its concentration, the function of (a4))(f32)2 receptors. Mutational studies in combination with receptor modelling showed that the structural deternlinants responsible for 2n2 + potentiation and 2n2 + inhibition reside at the a4-a4 and at the f32-a4 subunit interfaces, respectively. Finally, to circumvent any uncertainty on the stoichiometry of a4J32 expressed in Xenopus oocytes or other heterologous systems, the expression of the two a4f32 receptor stoichiometries was constrained by means of subunit concatenation. In Xenopus oocytes concatenated receptors have produced functional expression levels comparable to those obtained by non-linked subunits. The data presented here suggest that subunit concatenation does not alter the receptor properties, thus making pentameric concatenated constructs a powerful tool for addressing the fine study of the structure and function of receptors.
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Sun, Jiayin. "The Structural Basis for Lipid-Dependent Uncoupling of the Nicotinic Acetylcholine Receptor." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35642.
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In lipid membranes lacking activating lipids, the nicotinic acetylcholine receptor adopts an uncoupled conformation that binds ligand, but does not transition into an open conformation. Understanding the mechanisms of lipid-dependent uncoupling is essential to understanding lipid-nAChR interactions, which may be implicated in pathological conditions such as nicotine addition. Here, I tested two structural features of a proposed uncoupling method to elucidate the mechanism of lipid-dependent uncoupling. First, infrared measurements and electrophysiological characterization performed in prokaryotic homologues indicate that lipid sensitivity is largely controlled by the most peripheral α-helix in the transmembrane domain, M4. My data show that tighter association of M4 with the adjacent M1 and M3 transmembrane α-helices decreases a receptor’s propensity to adopt a lipid-dependent uncoupled conformation. Second, I indirectly tested the hypothesis that uncoupling results from a conformational change at the extracellular/transmembrane domain interface that leads to an increased separation between the two domains and ultimately to a constriction of the channel pore. Finally, biophysical studies presented in this dissertation shed light on the complex binding of a number of non-competitive channel blockers to the nicotinic acetylcholine receptor channel pore in both the resting and desensitized states. The data provide further insight into the structural rearrangements that occur upon uncoupling of ligand binding and gating in the nicotinic acetylcholine receptor.
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Montpetit, Madeleine R. "Characterization of multiple conductance states in a nicotinic acetylcholine receptor/channel preparation." Thesis, University of Ottawa (Canada), 1986. http://hdl.handle.net/10393/11054.
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The acetylcholine-activated channel of vertebrate skeletal muscle, as manifested in cultured developing cells, is able to adopt more than one conductance state. This thesis reviews the evidence for such multiple conductance channels and presents single channel data suggesting that subconductances represent discrete, allosterically-activated channel conformations. Since the amplitude of subconductance openings does not depend on agonist size or valence, the possibility that subconductances occur during partial occlusion of the channel by agonist molecules was ruled out. The conductances found for each event type are as follows; M Type = 40pS, S1 = 13pS and S2 = 7pS. While agonists do not determine conductance of the AChR, different agonists have differential abilities at stabilizing the sublevel openings. The kinetic analysis of open time histograms for the various conductance states reveals that the full channel openings are made up of two distinct kinetic open states; one of which is agonist-dependent and the other agonist-independent. In general, the subconductances S1 and S2 both displayed openings with a single agonist-independent time constant. Bursting behavior in the G8 AChRs was observed when channels were activated by strong agonists.
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Vuong, Ngoc Quang. "Imaging the Nicotinic Acetylcholine Receptor in Reconstituted Membranes by Atomic Force Microscopy." Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28592.
Full textAbstract:
Several recent studies have suggested that the nicotinic acetylcholine receptor (nAChR) segregates lipids into domains in reconstituted bilayers. These studies, however, lack direct evidence (e.g., microscopic images) to show domain formation. Atomic force microscopy (AFM) has been used extensively to image both lipid domains and proteins in membranes, but has not been applied extensively to reconstituted membrane proteins due to the lack of available protocols for preparing suitable planar bilayers on AFM supports. The aim of the work presented in this thesis was to image the nAChR in planar reconstituted membranes by AFM. I developed a novel method for reconstituting the nAChR in POPC (POPC-nAChR) to generate vesicles with high lipid-to-protein (L:P) ratios (i.e., greater than 100:1 w/w). Freeze-thaw cycles are required to improve vesicle homogeneity. The high L:P vesicles must also be isolated from protein-free vesicles using sucrose density gradients. Finally, the preparation of planar bilayers from the high L:P ratio proteoliposomes requires an appropriate sample load and incubation time on a defined area of mica surface (the solid AFM support) and an appropriate level of calcium. AFM images of a POPC-nAChR bilayers show a number of features that protrude out of the bilayer with an average height of 3 nm and diameter of 4 -- 9 nm, which is appropriate for the dimensions of the cytoplasmic side of the nAChR. My results thus represent the first AFM images of the nAChR in a reconstituted membrane environment. Now that the key parameters governing nAChR reconstitution and planar bilayer preparation for AFM imaging are understood, they will undoubtedly be useful for reconstituting and imaging the nAChR in more complex bilayers.
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Ryan, Stephen E. "Conformational effects of lipids and local anesthetics on the nicotinic acetylcholine receptor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/NQ57064.pdf.
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Fraser, David Michael. "Modulation of lipid-protein interactions of the nicotinic acetylcholine receptor by anaesthetics." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.253292.
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Cooper, Sandra Terese. "Cell specific folding of neuronal nicotinic acetylcholine receptor a7 and a8 subunits." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299622.
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Lee, Robert Hugh. "Alpha7 Nicotinic Acetylcholine Receptor: Novel Role in Macrophage Survival and Murine Atherosclerosis." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1418050832.
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Taylor, James H. (James Harvey) 1970. "Nicotinic Acetylcholine Receptor α3 mRNA in Rat Visual System After Monocular Deprivation." Thesis, University of North Texas, 1997. https://digital.library.unt.edu/ark:/67531/metadc278900/.
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In situ hybridization was used to examine effects of monocular enucleation on
nicotinic acetylcholine receptor subunit cc3 mRNA in the rat dLGNand visual cortex. After 28 days postoperative, there were no significant differences in α3 mRNA density between the contralateral (deprived) and ipsilateral (non-deprived) sides. The lack of obvious effects of visual deprivation on α3 mRNA density suggests that other factors, possibly intrinsic to dLGNand visual cortex, govern the postnatal expression of α3 mRNA.
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Molles, Brian Edward. "Probing the structure of the muscle nicotinic acetylcholine receptor with waglerin peptides /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2001. http://wwwlib.umi.com/cr/ucsd/fullcit?p3022198.
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Milholland, Rebecca. "L-type calcium channels mediate nicotinic acetylcholine receptor aggregation on cultured myotubes." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/280370.
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In this dissertation, I have presented new information on several aspects of the signaling pathway responsible for the clustering of AChRs on muscle cells. First, I have shown that activation of L-CaChs is both necessary for agrin induced clustering of AChRs and sufficient to stimulate AChR clustering even in the absence of agrin. Additionally, I have shown that activation of AChRs causes their own clustering by influencing the activity of L-CaChs. I have also shown that neither AChRs nor L-CaChs play a role in MuSK activation or AChR beta subunit phosphorylation suggesting that the role of AChR and L-CaCh is downstream of MuSK activation and phosphorylation of the AChR beta subunit in the signaling cascade that leads to the aggregation of AChRs. Finally, I have shown that calcium induced clustering and phosphorylation of AChRs require LCaCh activation. These data suggested that although L-CaCh activation is insufficient to cause AChR beta subunit phosphorylation L-CaCh may modulate an intermediate step between MuSK activation and AChR phosphorylation. These data therefore support the hypothesis that L-CaCh activation delivers extracellular calcium to the intracellular machinery that regulates AChR clustering. Furthermore, these data establish the position of L-CaChs in the signaling hierarchy responsible for AChR clustering as being downstream of or parallel to both MuSK activation and AChR phosphorylation in the signaling cascade behind AChR clustering. The data presented in this paper begin to provide an integrated view of NMJ formation in which neuromuscular transmission, calcium signaling, and signaling cascades mediated by neurotrophic factors act in concert to regulate the localization of synaptic molecules to junctional regions of the muscle fiber. Many questions remain, however, regarding the events downstream of MuSK and L-CaCh activation.
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Luo, Yujia. "Analgesic Effect of Nicotine and Exploration of Binding Sites for α4β2 Nicotinic Acetylcholine Receptor Positive Allosteric Modulators." Thesis, The University of Sydney, 2023. https://hdl.handle.net/2123/30000.
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Pain is a leading cause of the global burden of disease and disability. Nicotinic acetylcholine receptors (nAChRs), especially the α4β2 subtype, are widely expressed throughout the central nervous system (CNS) and peripheral nervous system (PNS), making them critical analgesic targets for the development of novel drugs. In the early part of this thesis, the analgesic effects of nicotine were quantified through meta-analysis. Sixteen eligible articles, including five studies on pain tolerance (n = 210), five studies on pain threshold (n = 210), and 11 studies on pain scores (n = 1,148) were included. Prolonged laboratory-induced pain threshold and tolerance time and mild postoperative pain relief were observed in patients who received acute nicotine therapy. In addition, a network meta-analysis was conducted to compare the effectiveness of nicotine transdermal patch and nasal spray for postoperative pain. Eight eligible studies (n = 555) were included. The results showed that the nicotine transdermal patch had a stronger analgesic effect than the nicotine nasal spray did. In addition, the analgesic effect of the nicotine transdermal patch peaked at 1-1.5 hours. The results of our analysis revealed that nicotine produces a rapid analgesic effect in humans, but the long-term use of nicotine is likely to cause hyperalgesia. The side effects of nicotine have been reported in several studies, likely due to the absence of nAChR subtype selectivity of nicotine. This limits the clinical applications of nicotine. Consequently, there is a need to study nAChR-targeted compounds with high receptor selectivity in order to replace nicotine.
Pharmacological studies of subtype-selective positive allosteric modulators (PAMs) with regard to the α4β2 nAChR (the most abundant subtype of nAChR in humans) are described in the following part of this thesis. The α4β2 nAChR is the most highly expressed subtype of nAChRs in the human brain, where it forms a high-affinity binding site for nicotine. NS206 was identified as a selective PAM of α4β2 nAChRs. LY2087101 and dFBr have pharmacological profiles similar to those of NS206, suggesting that they may bind to the same binding site(s) in wild-type (α4)3(β2)2 nAChRs. Notably, the α4 and β2 subunits assembled into a mixture of (α4)2(β2)3 (high-affinity) and (α4)3(β2)2 (low-affinity) nAChRs in Xenopus laevis oocytes, and the two stoichiometries differ significantly in their functional and pharmacological properties, for instance, the (α4)3(β2)2 nAChR exhibits a biphasic ACh concentration-response relationship, whereas the (α4)2(β2)3 nAChR exhibits a monophasic ACh concentration-response relationship. Therefore, the pharmacological studies on NS206 have been conducted on both stoichiometries. A uniform population of either (α4)3(β2)2 or (α4)2(β2)3 nAChRs in oocytes was obtained by injecting with α4 and β2 cRNA at ratios of 10:1 and 1:4, respectively. The addition of NS206 increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs (2.04 ± 0.64 vs. 0.88 ± 0.13, and 2.61 ± 0.42 vs. 0.96 ± 0.03, respectively), which is a statistically significant difference. However, a change in EC50s of the ACh concentration-response curves was not observed. In addition, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups.
To determine the binding sites of NS206, amino acid substitutions of phenylalanine-substituted leucine at position 256 (α4L256F) and leucine-substituted phenylalanine at position 316 (α4F316L) in the transmembrane domain (TMD) of the α4-subunit were performed using site-directed mutagenesis technique. Compared with the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents [(α4)3(β2)2: 6.91 ± 0.57 vs. 6.23 ± 4.35 and 0.84 ± 1.18, respectively; (α4)2(β2)3: 4.14 ± 0.38 vs. 2.37 ± 0.1 and 0.94 ± 0.14, respectively]. In addition, the NS206 potency showed a right-shifted trend for the mutated nAChRs. These results indicate that α4L256 and α4F316 are potential binding sites for NS206 in α4β2 nAChRs.
In summary, this thesis first discussed the involvement of nAChRs in pain by determining the analgesic effect of nicotine on laboratory-induced pain and acute postoperative pain. Furthermore, we compared the analgesic effects of two common clinical nicotine dosage forms, the nicotine transdermal patch and nasal spray, and determined the time of the peak analgesic effect. We found that the analgesic effect of the nicotine transdermal patch was better than that of nicotine nasal spray, peaking at approximately 1-1.5 hours. This thesis studied the pharmacological profiles of NS206 and investigated the binding interactions of NS206, LY2087101, and dFBr. We found that adding NS206 hardly changed the EC50s of ACh concentration-response curves, but significantly increased the height of peak responses at (α4)3(β2)2 and (α4)2(β2)3 nAChRs. Furthermore, no significant additive effect or change in potency was observed when comparing the NS206 + LY2087101 and NS206 + dFBr groups to the NS206, LY2087101 or dFBr groups. Finally, we identified two potential binding sites of NS206. Compared to the wild-type (α4)3(β2)2 and (α4)2(β2)3 nAChRs, the mutated receptors with amino acid substitutions α4L256F and α4F316L significantly reduced NS206 potentiated maximum responses of ACh-evoked currents. Furthermore, the NS206 potency showed a right-shifted trend for mutated nAChRs. These results indicate that α4L256 and α4F316 are the potential binding sites of NS206 on the α4β2 nAChRs. Combined with molecular modelling these results may facilitate delineation of the binding mode of the studied compounds and may thereby pave the way for the future rational design and discovery of novel modulators.
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Geng, Lin. "Visualization of nicotinic acetylcholine receptor trafficking with quantum dots in xenopus muscle cells /." View abstract or full-text, 2006. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202006%20GENG.
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Méthot, Nathalie. "Secondary structure of the channel forming transmembrane segments from the nicotinic acetylcholine receptor." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0020/NQ46533.pdf.
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Mozayan, Mansoor. "Modulation of sympathetic [alpha]7-nicotinic acetylcholine receptor by cholinesterase inhibitors and statins /." Available to subscribers only, 2006. http://proquest.umi.com/pqdweb?did=1240700261&sid=2&Fmt=2&clientId=1509&RQT=309&VName=PQD.
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Moss, Stephen James. "Cloning and expression of the genes encoding the chick muscle nicotinic acetylcholine receptor." Thesis, Imperial College London, 1989. http://hdl.handle.net/10044/1/47576.
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Ziviani, Elena. "Long term effect of nicotinic acetylcholine receptor activation and downstream signalling in neurons." Thesis, University of Leicester, 2005. http://hdl.handle.net/2381/30777.
Full textAbstract:
Nicotinic acetylcholine receptors (nAChRs) are widely present in the central nervous system (CNS). However, their cellular functions and physiological significance are not yet fully understood. Recently, in vivo and in vitro studies have suggested a possible neuroprotective role for nAChRs. Exposure to nicotine, an agonist at nAChRs, has been shown to protect from neurotoxicity induced by NMDA, glutamate and potassium withdrawal, and to prevent beta-amyloid induced neurotoxicity. Furthermore, stimulation of nAChRs has been shown to delay the ageing process of nigrostriatal neurons, increase neurotrophic factor levels and up-regulate the expression of NGF receptors in the brain. These observations may provide a basis for the findings that cigarette smoking is negatively correlated with Parkinson's disease and positively correlated with the delayed onset of Alzheimer's disease (AD). In conjunction with this, nicotine has been shown to affect a wide variety of biological functions including gene expression.;As nAChRs are calcium permeable channels, we have focused this current study on the effect of nicotine on the regulation of cellular proteins responsible for calcium-mobilisation, -transportation and -buffering. We found that in cortical neurons treated with 10microM nicotine for 24 h there is a selective up-regulation of ryanodine receptor 2 (RyR2), a calcium release channel present in the endoplasmic reticulum. In contrast, the expression of the other calcium-transporting and calcium-binding proteins tested were not affected. In nicotine treated cortical neurons, the increase RyR number was mirrored by a significant modification of the calcium response upon ryanodine stimulation.;In conclusion, our data show for the first time the effect of nicotine on the expression of proteins involved in calcium homeostasis.
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Young, G. T. "Molecular characterisation of nicotinic acetylcholine receptor subtypes : interactions with agonists and allosteric modulators." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/17315/.
Full textAbstract:
Nicotinic acetylcholine receptors (nAChRs) are members of the ligand-gated ion channel family. They are also members of the sub-family of Cys-loop ligandgated ion channels, which includes closely related receptors for the neurotransmitters γ-aminobutyric acid (GABA), glycine, and 5- hydroxytryptamine (serotonin). There is a wealth of evidence demonstrating that nAChRs are involved in mediating fast excitatory transmission, and also that they modulate the release of other neurotransmitters. They have been implicated in several neurological disorders such as Alzheimer’s disease, Parkinson’s disease, and schizophrenia, and also mediate the effects of nicotine associated with tobacco smoking. They are therefore viewed as potentially useful targets for the development of therapeutic drugs. In addition to the endogenous neurotransmitter acetylcholine, nAChRs are targets for a diverse collection of naturally occurring ligands. These include agonists and antagonists isolated from plants, freshwater algae, marine worms, frogs, the venoms of snakes and predatory marine snails. An increasing collection of synthetic ligands have also been produced which act upon the nAChR. The aim of work presented in this thesis is to investigate recently developed ligands of the nAChR and to characterise their action. Work is presented aimed at characterising TMAQ, a β4-selective nAChR agonist. Strikingly, this agonist exhibits activity only upon nAChRs containing the human β4 subunit whilst showing no activity on receptors containing the rat β4 subunit. Evidence is presented which, using hybrid receptors and chimeric subunits, identifies the extracellular portion of the human β4 subunit as being critical to this species-selectivity. More specifically, the work demonstrates that a region of the extracellular domain (loop D) of the β4 subunit is responsible for this species selectivity. Additionally, it is shown that, although acting as an agonist of human β4-containing nAChRs, TMAQ acts as an antagonist of rat β4-containing nAChRs. A homology model of the β4 subunit is also presented which identifies the location of the important residues; rationale is provided for their effects. Work is also reported which characterises positive allosteric modulators of α7 nAChRs, another class of nAChR ligand. Two classes of positive allosteric modulators are examined utilising prototypes from each class. It was found that type I positive allosteric modulators, such as LY-2087101, increase the magnitude of peak response to agonists. Type II positive allosteric modulators, such as PNU- 120596, not only increase the magnitude of peak response to agonist but also slow the receptor’s rate of desensitisation. Chimeric α7/5HT3A subunits were used to identify domains crucial to the site of action for PNU-120596; the first three transmembrane domains were found to be essential. Mutation of individual residues within the α7 transmembrane domains into the equivalent amino acids in the 5HT3A subunit identified several key residues. Homology modelling and computational docking of allosteric modulators helped to support the theory that these residues line the binding site for PNU-120596 and LY-2087101. Due to this site’s similarity to modulatory sites found in other closely related receptors it is argued that it represents a conserved modulatory site across the Cys-loop family of receptors.
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Marvin, Jessica Manichanh Catherine. "The levamisole sensitive nicotinic acetylcholine receptor of the potato cyst nematode Globodera pallida." Thesis, University of Leeds, 2015. http://etheses.whiterose.ac.uk/11982/.
Full textAbstract:
The potato cyst nematode Globodera pallida costs the UK potato industry over £50 million per annum. In order to invade a host plant, the infective J2 stage must hatch from eggs within the soil and migrate towards the root system. Orthologues of Caenorhabditis elegans genes involved in neurotransmission were identified in the G. pallida and G. rostochiensis genome assemblies. The complement of cys loop ligand gated ion channel genes was distinct compared to C. elegans and other parasitic nematodes. Orthologues of genes encoding subunits which comprise the C. elegans levamisole sensitive nicotinic acetylcholine receptor (cel-lev 1, cel-lev 8, cel-unc 29, cel-unc 63 and cel-unc 38) were searched for, and cel-lev 1 and cel-lev 8 orthologues were absent in both Globodera spp. Two orthologues were identified for cel-unc 29 and cel-unc 38. This suggested that the composition of the G. pallida L nAChR may differ. The use of C. elegans as a heterologous system to study the expression pattern of G. pallida nAChR genes was explored. GFP expressing lines were created using promoter regions of gpa acr 2 and gpa unc 63. Expression was observed in the ventral nerve cord and nerve ring for pgpa-acr 2. Expression of pgpa-unc 63 was variable, but was found in the head and tail region and along the ventral side of the body. The impact of this distinct complement of cys loop subunits on anthelmintic sensitivity was demonstrated by the increased resistance of both G. pallida and G. rostochiensis J2s to levamisole. The EC50 of G. pallida and G. rostochiensis was 19.7 mM and 5.6 mM respectively, compared to the EC50 of 9 µM for C. elegans, representing a 500 – 2000 fold increase in levamisole resistance. This increased resistance to levamisole was associated with an orthologue of cel-unc 38 identified in G. pallida, gpa unc 38.1. Rescue of C. elegans unc 38(x20) mutants with gpa unc 38.1 restores normal movement suggesting a functional reconstitution of the L nAChR, but full sensitivity to levamisole is not restored. Gpa unc 38.1 was expressed with the remaining four subunits from C. elegans in Xenopus oocytes to produce a chimeric receptor. The EC50 of the response to acetylcholine and levamisole of the chimeric receptor and the native receptor was comparable and had similar opening responses to different agonists. Chimeric genes were created to analyse key motifs in gpa unc 38.1 that may affect receptor function and levamisole sensitivity. Gpa unc 38.1 was necessary for structural reformation of the receptor, but not acetylcholine binding. Removal or addition of a loop B glutamate residue, previously associated with levamisole sensitivity of Cel UNC 38, did not affect levamisole sensitivity of Cel-UNC 38 or Gpa UNC 38.1. An amino acid change (I > M) in TM2 of Cel-UNC 38 increased levamisole sensitivity and basal thrashing rate. The reciprocal change (M > I) in Gpa UNC 38.1 comprised basal thrashing rescue. The basis of increased levamisole resistance of gpa unc 38.1 was not identified, as all gpa unc 38.1 chimeric genes retained a higher resistance to levamisole than cel-unc 38. This works reveals that the nAChRs of plant parasitic nematodes have distinct pharmacological characteristics.
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Magrone, P. "Design, synthesis and pharmacology of novel ligands targeting neuronal nicotinic acetylcholine receptor subtypes." Doctoral thesis, Università degli Studi di Milano, 2008. http://hdl.handle.net/2434/152407.
Full textAbstract:
Neuronal nicotinic acetylcholine receptors (nAChRs) make up a family of pentameric ligand-gated ion channels which are formed by combinations of alpha and beta subunits or exist as homopentamers, in the cases of α-7, α-8, and α-9 receptors, which are inhibited by α-bungarotoxin. To date, nine α (α-2-α-10) and three β(β2-β4) isoforms have been characterized, though only a relatively small subset of combinations generates functionally and physiologically relevant channels. Nicotinic receptors are widely distributed in the brain, where they primarily modulate the release of other neurotransmitters and, to a lesser extent, mediate synaptic transmission. Neuronal nAChRs are involved in various processes such as cognition, learning and memory, cerebral blood flow and metabolism, as well as an array of pathological conditions such as Alzheimer’s and Parkinson’s diseases, mild cognitive impairment (MCI), schizophrenia, epilepsy, Tourette’s syndrome, anxiety, depression, attention-deficit hyperactivity disorder (ADHD), and nicotine addiction. The heteromeric α4β2 and the homomeric α7 nAChR subtypes represent the most relevant biological targets in view of potential therapeutic applications for the above cited pathologies.
In this study, the group of novel stereoisomeric Δ2-isoxazolines 1a-1f and 2a-f, structurally related to the α4β2 selective nicotinic agonist ABT-418, has been prepared and tested at neuronal α4β2 and α7 nAChR subtypes. Moreover, (-)-Cytisine 3, a natural selective α4β2 ligand, was taken as model compound to synthesize the group of 4-substituted derivatives 4-8.The two series of derivates were synthesized by taking advantage of a 1,3-dipolar or a 1,6-intramolecular cycloaddition processes.On the other hand, as an extension of a previous research project, we prepared a group of novel derivatives characterized by a spirocylic junction between the quinuclidine ring and the Δ2-isoxazoline (8a-g and 9a-g) or the isoxazolidin-3-one (10a-b) moieties. Some of the compounds under study behaved as potent and selective α7 nAChR agonists/partial agonists and were further investigated in in vitro and in vivo tests.
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Domville, Jaimee Allison. "Mapping the Allosteric Pathway Leading from a Mutation in the Nicotinic Acetylcholine Receptor to a Congenital Myasthenic Syndrome." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/37037.
Full textAbstract:
The peripheral and highly lipid-exposed M4 α-helix, although distant from the agonist binding site, channel gate, and other important gating structures, is involved in modulating function of the nicotinic acetylcholine receptor. M4 "senses" changes in the surrounding lipid environment and may consequently affect receptor function by altering specific interactions between the M4 C-terminus and the Cys-loop. An example of this lipid sensing ability is demonstrated by a lipid-facing Cys418 to Trp substitution on αM4 (αM4 C418W) of the muscle-type receptor, which subtly alters protein-lipid interactions and potentiates channel function 16-fold, leading to a slow-channel congenital myasthenic syndrome. Through the use of mutational studies and mutant cycle analysis, I determine that, contrary to previous studies, M4–Cys-loop interactions are not critical to wild-type muscle-type receptor function, nor are they involved in C418W-induced potentiation. Instead, C418W potentiates channel activity by enhancing local M4-M1 interactions mediated by three polar side-chains, which are absolutely critical to potentiation. I show that altered M4-M1 interactions are ultimately translated to two important gating structures, which work in tandem to stabilize the open conformation of the receptor. These studies highlight how altered protein-lipid interactions can affect channel function and contribute to our understanding of the underlying gating mechanism of the muscle-type receptor.
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MELIS, MIRIAM. "Fatty acid ethanolamides pinpoint nicotinic receptors and modulate neuronal excitability through nuclear receptor PPARα." Doctoral thesis, Università degli Studi di Cagliari, 2011. http://hdl.handle.net/11584/266354.
Full textAbstract:
SPECIFIC AIMS OF THE STUDY
1) To investigate whether FAEs can suppress nicotine-induced stimulation of VTA dopamine
(DA) neuron firing rate through the peroxisome-proliferator-activated receptor-α (PPARα)
and to elucidate their mechanism of action in an in vitro brain slice preparation.
2) To determine whether there is an interaction between PPARα and nAChRs in VTA DA
cells: particularly, to characterize the postsynaptic effects of PPARα modulation on VTA
DA neurons, and to examine the contribution of nAChRs in the effects.
3) To study whether PPARα modulation of nAChRs in VTA DA neurons might confer DA
cells to access distinct firing patterns and/or to change their firing frequency both in vivo
and in vitro.
4) To examine the physiological relevance of PPARα modulation of nAChR stimulation
through behavioral analysis.