Dissertations / Theses on the topic 'M1 muscarinic acetylcholine receptor'
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1
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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Kim, Ju Young. "M1 muscarinic acetylcholine receptor regulation of endogenous transient receptor potential-canonical, subtype 6 (TRPC6) channels." Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1117570788.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xviii, 178 p.; also includes graphics. Includes bibliographical references (p. 163-178). Available online via OhioLINK's ETD Center
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Schroeder, Lee Frederick. "An in vivo biosensor for neurotransmitter release and In situ receptor activity acetylcholine and the M1 muscarinic receptor /." Diss., [La Jolla, Calif.] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3372798.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed October 20, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 100-120).
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Atkinson, Leone Sheila. "Development and characterization of herpes simplex virus type 1 vectors expressing m1 muscarinic acetylcholine receptors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp04/nq25012.pdf.
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Prihandoko, Rudi. "An investigation into the pharmacology and regulation of the M1, M3 and M4 muscarinic acetylcholine receptors." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/28575.
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Functional selectivity, which highlights the ability of ligands to differentially activate the signalling pathways linked to G protein-couple receptors (GPCRs) has provided an avenue for developing ligands with greater safety profiles. Pilocarpine (Pilo), a non-selective muscarinic acetylcholine receptor (mAChR) agonist has been shown to differentially activate G protein subtypes linked to the M3 mAChR. In this study the pharmacology of Pilo was further investigated using a number of readouts. When compared to methacholine (MCh), a reference agonist, Pilo appeared to preferentially stimulate inositol phosphates production than global receptor phosphorylation. The ligand also appeared to preferentially promote phosphorylation of Ser412 at the third intracellular loop of the receptor than Ser577 at the C-terminal tail. This differential phosphorylation may be linked to the fact that these residues are phosphorylated by distinct protein kinases. However, such preferential phosphorylation was not evident at the mutant M3 RASSL receptor that was engineered to respond to Clozapine-N-oxide (CNO). This mutant receptor was phosphorylated in response to CNO stimulation in a similar manner as the wild-type M3 mAChR responding to ACh. Allosteric modulation has been considered an attractive approach to selectively target GPCR subtypes for multiple disease indications. BQCA and LY2033298 have been shown to act allosterically at the M1 and M4 mAChR, respectively. In this study, we provided evidence that BQCA is probe dependent and the compound is more potent as an affinity modulator of ACh than Pilo. However BQCA did not significantly potentiate the phosphorylation state of the M1 mAChR following stimulation with a sub-maximal concentration of ACh. Similar results were obtained for LY2033298 at the M4 mAChR which suggest that allosteric modulators do not promote a receptor conformation that increases the accessibility of phosphorylation sites to protein kinases.
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Ward, Stuart David Charles. "The role of residues Tyr381 to Val387, in transmembrane domain six of the rat M1 muscarinic acetylcholine receptor, in agonist binding and receptor activation." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390625.
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Buffat, Maxine Guy Patrick. "Synthesis of selective M1 muscarinic receptor agonists." Thesis, University of Manchester, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.488787.
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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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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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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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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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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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Boxall, Donna Kirsty. "Muscarinic acetylcholine receptor signalling in model cells and smooth muscle." Thesis, University of Leicester, 1998. http://hdl.handle.net/2381/29959.
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In this Thesis, experiments are described in which the signalling of M2- and M3-mACh receptors as homogeneous receptor populations and as a co-expressed population in mammalian cell-lines have been studied. These experiments set out to investigate the hypothesis that M2-mACh receptors can influence signalling by M3-mACh receptors via 'cross-talk' at the level of second messenger signalling. This may have particular significance with respect to mACh receptors in smooth muscle. In many smooth muscle tissues mACh receptor subtypes are co-expressed, with the major population often being M2-mACh receptors and the minor population being M3-mACh receptors. It is the M3-mACh receptors which have been implicated in the direct mediation of contraction via activation of phosphoinositide metabolism. Whilst it is known that M2-mACh receptors signal via inhibition of adenylyl cyclase, it is not known whether M2-mACh receptors can modulate signalling via M3-mACh receptors to influence smooth muscle tone. By studying 'cross-talk' of these two receptors in a model cell system, this possibility can be evaluated. Chinese hamster ovary (CHO) cells expressing M2-, M3- or co-expressing M2- and M3- mACh receptors were characterised with respect to agonist-stimulation of inhibition of forskolin-stimulated cyclic AMP levels, and agonist-stimulation of inositol 1,4,5-trisphosphate (InsP3; time-course and concentration-dependence). These two responses have been shown to be representative of M2- and M3-mAaCh receptor-mediated responses, respectively. Whilst inhibition of cAMP production in CHO-SSLLM2 cells was pertussis toxin-sensitive and was blocked by tripitramine (30 nM), neither stimulation of cAAMP, nor InsP3 production in CHO-M3 cells was sensitive to either pretreatment. The studies indicate that cross-talk can occur between the signalling pathways of M2- and M3-mACh receptors in model cells. Further investigations to establish the mechanism of this cross-talk should include studies of signalling in a smooth muscle tissue which co-expresses these mACh receptors, to establish whether similar cross-talk occurs in a more physiological context.
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Schlador, Michael Lee. "Agonist-dependent regulation of muscarinic acetylcholine receptor expression and function /." Thesis, Connect to this title online; UW restricted, 2000. http://hdl.handle.net/1773/6251.
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Wylie, Paul. "Muscarinic acetylcholine receptor regulation of ERK and JNK in CHO cells." Thesis, University of Leicester, 2000. http://hdl.handle.net/2381/29924.
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Extracellular signal-regulated kinases (ERKs) and the c-Jun N-terminal kinases/stress- activated protein kinases (JNKs/SAPKs) are activated by an array of extracellular signals to mediate a variety of cellular responses e.g. mitogenesis, differentiation, hypertrophy and apoptosis. The study investigated the regulation of ERK and JNK by agonist-mediated stimulation of the human m2-AChR or m3-AChR stably expressed in CHO cells. Stimulation of both receptors dramatically activated ERK, although stimulation by the m3-AChR was more sustained. The m3-AChR is efficiently coupled to JNK activation, whereas the m2-AChR is not. Activation of JNK in CHO-m3 cells was delayed and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The dose-dependence for methacholine (MCh)-stimulated JNK activation by m3-AChR and ERK activation by both receptor subtypes were similar. Although pertussis toxin (PTX) had no effect on Ins(1,4,5)P3 accumulation in CHO-m3 cells, there was significant inhibition of agonist-induced ERK and JNK activation in CHO-m3 cells, suggesting that the m3-AChR was able to couple to Gi/o in addition to Gq. ERK activation was entirely PTX-sensitive in CHO-m2 cells. ERK activation in both cell types was shown to be independent of Ca2+. However, JNK activation by m3 receptors was shown to have both a Ca2+-depdnent and a Ca2+-independent component. PKC inhibition studies demonstrated a novel PKC- and an atypical PKC-component, but not a classical PKC-component in ERK activation in CHO-m2 cells, whereas, there appears to be a cPKC and an aPKC-component in CHO-m3 cells. In contrast to this, PKC appears to have an inhibitory role in m3-AChR-mediated JNK activation. The results presented demonstrate that the m2-AChR activate ERK and JNK via divergent mechanisms.
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Nuckels, Richard J. "Ontogeny of muscarinic acetylcholine receptor expression in the eyes of zebrafish /." View online, 2006. http://ecommons.txstate.edu/bioltad/2.
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Borroto, Escuela Dasiel Oscar. "Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signaling and regualation." Doctoral thesis, Universitat Politècnica de Catalunya, 2008. http://hdl.handle.net/10803/22678.
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Muscarinic acetylcholine receptors (mAChRs) have been shown to mediate various functions in the central and peripheral nervous systems. These include modulation of exocrine glandular secretion, vasodilatation and smooth muscle contraction, cell proliferation or survival, neural development and synaptic plasticity. mAChRs are activated by both endogenously produced acetylcholine and exogenously administered muscarinic compounds. Pharmacological, anatomical and molecular studies have demonstrated the existence of five muscarinic receptor subtypes, denoted as muscarinic M1, M2, M3, M4 and M5, which belong to class I family of heptahelical, transmembrane G-protein coupled receptors (GPCRs).
Each receptor subtypes are characterized by a distinct selectivity for heterotrimeric G protein coupling. Thus, M1, M3 and M5 are coupled to Gq/11 proteins and stimulate phospholipase C activity, resulting in the generation of the second messengers inositol (1,4,5)-trisphosphate (IP3) and diacylglycerol (DAG), the mobilization of intracellular Ca2+ and the activation of protein kinase C (PKC). On the other hand, M2 and M4 are coupled to Gi/0 proteins, which results in the inhibition of adenilate cyclase, as well as prolonging potassium channel, non-selective cation channel, and transient receptor potential channel opening. By means of this differential set of G protein partners, mAChRs can initiate distinct signalling pathways within a same cell in order to trigger diverse, even opposed, functional outcomes in response to the same stimuli. It has been proven as well that mAChRs regulate a baste network of signalling intermediates, including small GTPase Rho, phospholipase D, phosphoinositide-3 kinase, non-receptor kinases and mitogenactivated protein kinases.
Although the first proteins found to have functional interactions with mAChRs were, of course, G proteins, an increasing amount of evidence in the field suggests that this simplistic model defined as “one receptor -one G protein -one effector no longer exists. A great number of proteins have been identified as interacting with mAChRs, including GPCRs, kinases, and scaffolding proteins such as arrestin. Determining in part the signalling efficiency/specificity for mAChRs. Thus, receptors are now considered as complex signalling units, or signalosomes, that dynamically couple to multiple G proteins or other molecular entities or scaffold proteins in a temporally and spatially regulated manner, and even can form homodimers or heterodimers with distinct GPCRs or other non-GPCR membrane receptors, resulting in pharmacologically and functionally distinct receptor populations.
In this work “Human M3 muscarinic acetylcholine receptor protein-protein interactions: roles in receptor signalling and regulation”, it is discuss novel mAChRs interacting partners that link the receptors to alternative signalling pathways beyond G proteins. Emphases on explaining how mAChRs regulate signal transduction pathways mediated by these proteins, including receptor dimerization have been putting out.
It has been demonstrated by different approach (from resonance energy transfer to tandem affinity purification and mass spectrometry) the active role of interacting protein in mAChRs regulation and signalling. We shown that a particular complex is not necessarily of invariable composition, nor are all its building blocks uniquely associated with that specific complex. One complex may be the result not only of physical interaction between the receptor and the partners’ protein, but also of the participation of many non-“direct” associations resulting in the formation of a network that interconnects the receptor with a number of other pathways, determining receptor specificities. This allows us to address some fundamental questions concerning the importance of molecular mechanisms hidden behind the pharmacology properties for each receptor subtype.
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Akam, Elizabeth Claire. "The activation of guanine nucleotide binding proteins by muscarinic acetylcholine receptor subtypes." Thesis, University of Leicester, 1999. http://hdl.handle.net/2381/29919.
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Agonist-stimulation of human recombinant M1, M2, M3 and M4 receptors, expressed in Chinese hamster ovary cells, was investigated at the level of G protein activation. Functional responses were determined by a number of methods including [35S]-GTPS binding in membranes using both filtration-based and immunoprecipitation-based procedures: Ins(1,4,5)P3 accumulation and 45Ca2+ release from permeabilised cell suspensions; and cAMP accumulation in cell suspensions. M2 and M4 receptors, with equivalent expression levels in this recombinant system, were found only to couple to pertussis toxin-sensitive G proteins with near equal kinetics. Methacholine appeared equipotent when activating the total G protein complement through the M2 and M4 receptors, however, it appeared more potent when activating Gi3/o through the M2 compared to the M4 muscarinic receptor. Using equivalent expression levels of M1 and M3 receptors both the subtypes were found to couple to both pertussis toxin-sensitive and -insensitive G proteins. CHO-M1 and -M3 mediated Ins(1,4,5)P3 generation after pertussis toxin pre-treatment suggested the functional significance of coupling to multiple G protein classes may be in the stimulation of PLC by -subunits derived from Gi-like G proteins. The activation of Gq/11 through the M1 receptor subtype, after methacholine-stimulation, is faster, greater and more potent than that mediated by the M3 receptor subtype, suggesting that the intrinsic activity of the M1 subtype is greater than that of the M3 subtype. The 'partial' agonist pilocarpine also displayed very different G protein activation profiles after stimulation of M1, M2, M3 and M4 receptor subtypes, suggesting that agonists acting at different receptor subtypes may be capable of inducing relatively selective coupling of the occupied receptor to available G proteins. This study therefore concludes that muscarinic receptor subtypes display divergent G protein activation profiles after either 'full' or 'partial' agonist-stimulation.
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Jones, Kymry Thereasa. "The role of beta-arrestin in regulating the muscarinic acetylcholine type II receptor." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2007. http://hdl.handle.net/1853/24815.
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Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008.Committee Chair: Dr. Nael A. McCarty; Committee Co-Chair: Dr. Darrell Jackson; Committee Member: Dr. Alfred H. Merrill; Committee Member: Dr. Barbara D. Boyan; Committee Member: Dr. Harish Radhakrishna; Committee Member: Dr. Marion B. Sewer
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Romero, Fernández Wilber. "Posttranslational modifications of human M3 muscarinic acetylcholine receptor: zooming in its functional implications." Doctoral thesis, Universitat Politècnica de Catalunya, 2011. http://hdl.handle.net/10803/110544.
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The human M3 muscarinic acetylcholine receptor (M3R) regulates many important physiological roles in the central and peripheral nervous systems, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases, representing attractive potential pharmacological target for intervention. However, the lack of structural information on this receptor hampered the development of new potent antagonist with increased selectivity and lower side effects. Such structural information can be only achieved by means of experimental biophysical techniques, which require large quantities of pure receptor. Considering that under physiological conditions the expression of G-protein coupled receptors (GPCRs) is relatively low, optimization of the receptor overexpression is a pre-requisite for structural studies efforts to be performed. In addition, although is has well established that GPCR undergo post-translational and increase evidences support that these are tight links to receptor roles, little progress has been made in the post-translational modifications field in some GPCRs, such as the case of M3R.
In this study, we provide some strategies to improve muscarinic receptor heterologous expression in mammalian cells guaranteeing proper post-translational modifications. In addition, we have been able to extract high levels of functional receptor from COS-7 cells using a detergent combination tested, and to purify the receptor to near homogeneity-keeping the full wild type receptor properties- by means of different affinity purification methods. Regarding the post-translational modifications studied, our findings provide the first evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution and localization, as well as in cell integrity. Furthermore, our data reveal a role for palmitoylation in determining M3R residence within lipid raft, as well as in receptor internalization and down-regulationEl receptor muscarínico de acetilcolina subtipo M3 humano (M3R) regula importantes funciones en el sistema nervioso central y periférico, y está implicado en la fisiopatología de varias enfermedades neurodegenerativas y autoinmunes, lo que representa una atractiva diana terapéutica para la intervención farmacológica. Sin embargo, la falta de la información estructural sobre este receptor obstaculizado el desarrollo de nuevos y potente fármacos de gran selectividad y bajo efecto secundario. Tal información estructural, puede lograrse por medio de la experimentación con técnicas biofísicas que requieren grandes cantidades de receptor puro. Teniendo en cuenta que en condiciones fisiológicas la expresión de receptores acoplados a proteínas G (GPCR) es baja, la sobreproducción del receptor es un pre-requisito para que los estudios estructurales puedan ser realizados. Además, aunque se ha establecido que los GPCR sufren modificaciones post-translationales y que en los últimos años un significante número de reportes sugieren que estas modificaciones están estrechamente vinculadas a las funciones del receptor, poco se ha avanzado en el estudio de estas modificaciones en el campo de algunos GPCRs, como es el caso de M3R. En este estudio, nosotros describimos algunas estrategias para mejorar la expresión de los receptores muscarínicos en células de mamíferos garantizando unas correctas modificaciones post-translacionales. Además, hemos sido capaces de extraer altos niveles de receptor funcional a partir de células COS-7 con una combinación de detergentes, purificamos el receptor M3R cerca de la homogeneidad, mantenimiento de la totalidad de las propiedades biológicas encontradas en el receptor silvestre. En relación a las modificaciones post-translationales estudiadas, nuestros resultados proporcionan la primera evidencia del papel crítico de las cadenas de N-glicanos en la determinación de la localización de estos receptores, así como en la integridad celular. Además, nuestros datos revelan un importante papel de las modificaciones lipídicas de M3R en relación a la distribución del receptor en microdominios resistente a detergentes, así como en la regulación del receptor. En resumen, las estrategias utilizadas pueden contribuir al incremento de la expresión M3R. De esta forma los esfuerzos para la purificación del receptor a gran escala pueden ser iniciados. Para ellos, nosotros revelamos una posible estrategia. Además, proponemos los posibles sitios de N-glicosilación y S-acilación en el M3R expresado en células COS-7, y proporcionamos evidencias experimentales que avalan la implicación funcional de estas modificaciones en el papel del receptor.
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Hornigold, David Charles. "Molecular studies of cross-talk between M2 and M3 muscarinic acetylcholine receptor subtypes." Thesis, University of Leicester, 2001. http://hdl.handle.net/2381/29927.
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Many cell-types in the body express mixed populations of muscarinic acetylcholine (mACh) receptors. For example, various types of smooth muscle express both M2 and M3 mACh receptors. While M3 mACh receptor sub-population is implicated in pharmacomechanical coupling in smooth muscle, the role of the M2 mACh receptor remains unclear. The aim of the studies described here was to investigate if there is 'cross-talk' between the signalling pathways regulated by M2 and M3 mACh receptors, co-expressed in either model (CHO) cells, or a smooth muscle tissue preparation. Based on [3H]-NMS radioligand binding, cyclic AMP and inositol 1,4,5-trisphosphate (IP3) mass measurements, and single cell Ca2+-imaging experiments, stable and functional co-expression of M2 and M3 mACh receptors was demonstrated in two CHO-m2m3 cell lines. Initial experiments provided little evidence for M2/M3 mACh receptor cross-talk in CHO-m2m3 cells, as co-stimulation of the M2 receptor had no discernible effect on M3 receptor-mediated phospholipase. C activation, assessed at the level of IP3 and [Ca2+]i. Although both M2 and M3 mACh receptor stimulation modulated adenylyl cyclase activity, the biphasic modulation of cyclic AMP concentration in the CHO-m2m3 cells could be explained as additivity between the responses observed in CHO-m2 and -m3 cells. M2 and M3 mACh receptor stimulation caused essentially opposite effects on cell proliferation, assessed by measuring [3H]-thymidine incorporation into DNA, in CHO-m2 and CHO-m3 cells. In CHO-m2m3 cells, the inhibitory M3 mACh receptor-mediated effect appeared to dominate with respect to cell growth, however, a proliferative M2 mACh receptor-mediated effect could be 'unmasked' using the subtype-selective mACh receptor antagonist, darifenacin.
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McKinnon, Lise Anne. "Developmental regulation of muscarinic acetylcholine receptor expression in embryonic chick heart and retina /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6263.
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Bee, Stephen. "Alanine-scanning mutagenesis of transmembrane helix 2 of the rat Mâ‚ muscarinic acetylcholine receptor." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.397428.
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Sandiford, Simone Laura. "Interaction of the G Beta Sub Five-RGS7 Complex with the Muscarinic Acetylcholine M3 Receptor." Scholarly Repository, 2009. http://scholarlyrepository.miami.edu/oa_dissertations/311.
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Regulators of G protein signaling (RGS) are a diverse group of proteins, which play a fundamental role in modulation of G protein coupled receptor signal transduction. RGS proteins are primarily known as GTPase activating proteins (GAPs) for Gá subunits. In addition to the RGS domain, which is responsible for GAP activity, most RGS proteins also contain other structural motifs. The R7 family of RGS proteins for example, which consists of RGS-6, 7, 9 and 11 gene products, also contains DEP, DHEX and GGL domains. All R7 RGS proteins are obligatory binding partners with G protein beta subunit, G beta sub five, which binds to the GGL domain. In my dissertation work, I provide insights into significance of the multi-domain architecture of G beta sub five-RGS7. I have identified a novel intramolecular interaction within the G beta sub five-RGS7 complex; between the DEP domain of RGS7 and G beta sub five subunit. My experimental evidence supports the idea that G beta sub five-RGS7 can exist in at least two hypothetical conformations: "closed" where the DEP domain and G beta sub five subunit are bound to each other, and "open" where DEP and G beta sub five are not interacting, and as a result both these proteins can associate with other binding partners. My results indicate that in its "open" conformation, G beta sub five-RGS7 can selectively inhibit calcium mobilization elicited by stimulated muscarinic acetylcholine receptor type 3 (M3R). This inhibition is mediated by direct interaction between the third intracellular loop of M3R and the DEP domain of RGS7. In addition to the effect on M3R signaling, I observed that the G beta sub five-RGS7 complex redistributes from the cytosol to endocytic vesicles in an M3R-specific manner. These results identify a novel molecular mechanism that can impart receptor-subtype selectivity on signal transduction via G protein-coupled receptors. Lastly, I have identified a small group of compounds that inhibits the DEP-G beta sub five interaction. These compounds may serve as starting points for design of G beta sub five-RGS7 modulators in the future.
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Wang, Alice Wu. "Muscarinic acetylcholine receptor heterogeneity in the central nervous system of the tobacco hornworm, Manduca sexta /." Thesis, Connect to Dissertations & Theses @ Tufts University, 1998.
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Thesis (Ph.D.)--Tufts University, 1998.Adviser: Barry A. Trimmer. Submitted to the Dept. of Biology. Includes bibliographical references (leaves 92-105). Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Dowling, Mark R. "Exploring agonist dependency of receptor-G protein-coupling and constitutive activity at muscarinic acetylcholine receptors." Thesis, University of Leicester, 2004. http://hdl.handle.net/2381/29941.
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The effects of structurally diverse agonists were assessed on receptor-mediated activation of G-proteins using in Chinese hamster ovary cells stably expressing similar densities of M1 and M3 mACh receptors. Using a total [35S]-GTPgammaS binding protocol both receptor subtypes were shown to couple to both pertussis toxin-sensitive and -insensitive G-proteins. M1 mACh receptors coupled with greater potency and intrinsic efficacy than M3 receptors, indicating that in this cell-line that M1 mACh receptor may be more efficiently coupled to its complement of G-proteins. Total [36S]-GTPgammaS binding concentration-response curves for both receptor subtypes were shallow and, in the case of the M1 receptor, could be readily resolved into high and low affinity components. The heterogeneity of Galpha subunits activated was further investigated using an immunoprecipitation technique. This strategy revealed that agonist binding to M1 receptor caused the activation of a heterogeneous population of G-proteins, which was directly related to agonist efficacy. Full agonists were able to activate both Galphaq/11 and Galphail-3 subtypes, whereas partial agonists activated only the most efficiently coupled G-protein Galpha q/11. In contrast, the complement of G-proteins activated after M3 receptor stimulation was not related to the efficacy of the agonist and suggested that agonists may form active conformations of the M 3 receptor that possess different G-protein coupling profiles.;To investigate the G-protein coupling of M1 and M3 receptors further, homologous point mutations were introduced in both subtypes, which conferred agonist-independent constitutive activity. All of the antagonists tested were able to concentration-dependently inhibit basal [3H]-inositol phosphate accumulation and were therefore classified as 'inverse agonists'. However, subtle differences in the results obtained for different inverse agonists via different functional and binding readouts suggested the existence of more than one conformation of inactive receptor.
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Suharni. "Proteoliposome-based selection of a recombinant antibody fragment against the human M2 muscarinic acetylcholine receptor." Kyoto University, 2015. http://hdl.handle.net/2433/195961.
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Ragheb, Fadi. "The M3 muscarinic acetylcholine receptor mediates p42mapk activation and c-fos mRNA expression in oligodendrocyte progenitors /." Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21627.
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Oligodendrocytes, the cells that produce and maintain myelin in the central nervous system (CNS), express several muscarinic acetylcholine (ACh) receptor (mAChR) subtypes. Carbachol (CCh), a stable analogue of ACh, stimulates various intracellular signals through mAChRs, including p42-mitogen-activated protein kinase (p42mapk) activation and c-fos gene expression. The present study examined the mAChR subtype(s) and the signaling pathway(s) involved in both responses using purified cultures of oligodendrocyte progenitors from newborn rats.The M3 mAChR selective antagonist 4-DAMP and its irreversible analogue 4-DAMP-mustard, but not pirenzepine (M1) or methoctramine (M2/M4), prevented p42mapk activation and c-fos mRNA expression. Pretreatment with a phospholipase C (PLC) inhibitor U73122 but not the inactive analogue U73343 blocked both responses, while the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect, suggesting the involvement of PLC but not PI3K.
In view of the fact that the M3 mAChR mediates p42mapk activation as well as c-fos mRNA expression, the MAPK Kinase (MEK) inhibitor PD 098059 was used to establish a link between both responses. PD 098059 pretreatment caused no significant attenuation in c-fos mRNA levels, demonstrating that this response may not be dependent on p42mapk activation. However, the phospholipase A2 (PLA2) inhibitors quinacrine and AACOCF3 abolished c-fos mRNA expression, implicating arachidonic acid and/or its bioactive eicosanoid metabolites.
In summary, these results demonstrate that in oligodendrocyte progenitors, the M3 mAChR is mediating both p42mapk activation and c-fos gene expression via PLC but not PI3K, whereby c-fos gene expression is not regulated by p42mapk but by PLA2.
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Garriga-Canut, Mireia. "Control of neuron specific gene expression : transcriptional regulation of the Mâ†1 muscarinic acetylcholine receptor gene." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367599.
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Peng, Joyce Yaochun Kennedy Mary B. "Structure and function prediction of human muscarinic acetylcholine receptor 1, cation-pi studies, and protein design /." Diss., Pasadena, Calif. : California Institute of Technology, 2005. http://resolver.caltech.edu/CaltechETD:etd-05312005-114949.
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Chirinda, Brian [Verfasser]. "The allosteric core region of the M2 muscarinic acetylcholine receptor : role for ligand selectivity and action / Brian Chirinda." Bonn : Universitäts- und Landesbibliothek Bonn, 2015. http://d-nb.info/107729042X/34.
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Schramm, Simon [Verfasser], and Michael [Gutachter] Decker. "Synthesis of Dualsteric Muscarinic M\(_1\) Acetylcholine Receptor Ligands and Neuroprotective Esters of Silibinin / Simon Schramm ; Gutachter: Michael Decker." Würzburg : Universität Würzburg, 2018. http://d-nb.info/1173088113/34.
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Gibson, Hayley. "The Effect of Muscarinic Modulators on Cilia Structure and Function." University of Toledo Health Science Campus / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=mco1499104909617105.
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Dickinson, Bryony. "An investigation into the mechanism of muscarinic acetylcholine receptor dependent long term depression in the CA1 region of the hippocampus." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.535163.
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Agnetta, Luca [Verfasser], Michael [Gutachter] Decker, and Pau [Gutachter] Gorostiza. "Novel Photoswitchable and Dualsteric Ligands Acting on Muscarinic Acetylcholine Receptors for Receptor Function Investigation / Luca Agnetta ; Gutachter: Michael Decker, Pau Gorostiza." Würzburg : Universität Würzburg, 2019. http://d-nb.info/1198682892/34.
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Risel, Philipp David [Verfasser], Peter [Akademischer Betreuer] Gmeiner, and Peter [Gutachter] Gmeiner. "Design and Synthesis of Novel Ligands for the M2 Muscarinic Acetylcholine Receptor / Philipp David Risel ; Gutachter: Peter Gmeiner ; Betreuer: Peter Gmeiner." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2020. http://d-nb.info/1222739569/34.
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Ragheb, Fadi. "The M3 muscarinic acetylcholine receptor mediates p42 [superscript]m [superscript]a [superscript]p [superscript]k activation and c-fos mRNA expression in oligodendrocyte progenitors." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape9/PQDD_0025/MQ50862.pdf.
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Heneghan, John F. "M1 Muscarinic Modulation of N-Type Calcium Channels: A Dissertation." eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/255.
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The influx of calcium through N-type calcium channels (N-current) affects a myriad of neuronal functions. These include the triggering of synaptic release of neurotransmitter, adjustment of membrane potential and changes in gene transcription. N-channels are highly modulated proteins, so that N-current is attenuated or potentiated in response to environmental changes. In turn, the modulation of N-current has a direct effect on the downstream events, making the N-channel a focal point in neural signaling, and its modulation a mechanism for short term plasticity.
The modulation of N-current by M1 muscarinic receptors (M1Rs) is of particular interest for several reasons. The M1R is instrumental in both cognition and memory formation as indicated by studies using either pharmacological agents aimed at M1Rs or knockout animals lacking M1Rs. Clinically, the M1R is an important target in the treatment of Alzheimer’s disease. Thus, like the N-channel, the M1R is an important element of neural signaling. Moreover, the stimulation of M1Rs affects N-current by through signaling pathways which despite being studied for decades, are not completely understood.
For my dissertation I have investigated of M1R signaling on N-current using electrophysiological recordings of N-current from freshly dissociated neurons and from HEK cells expressing N-channels and M1Rs. Asking how one receptor affects one type of calcium channel would seem to be a simple question. However, the answer has many facets. Since M1Rs have multiple downstream effects and N-channels are highly modulated proteins, stimulation of M1Rs initiates several different pathways which modulate N-current. This thesis aims to unravel some of the complexities of the interactions of two vital components of neuronal signaling. Here I present the results of studies elucidating three different actions of M1signaling of N-current modulation.
The first study I present here examines the effect of N-channel subunit composition on modulation of N-current. The stimulation of M1Rs in superior cervical ganglion (SCG) neurons elicits a distinct pattern of modulation; inhibiting N-current elicited by strong depolarizations and enhancing current elicited by lesser depolarizations. Thus M1Rs cause two simultaneous modulatory effects on N-current; increasing voltage sensitivity and decreasing overall conductance. I found the expression of the N-channel’s β subunit (CaVβ) determines the observed effect. Specifically when the isoform CaVβ2a is expressed M1 stimulation elicits enhancement without inhibition. Conversely, when CaVβ1b, CaVβ3, or CaVβ4 are expressed M1 stimulation elicits inhibition with out enhancement. These results fit a model in which both the enhancing and inhibiting effects of M1stimulation occur in all channels, but typically inhibition dominates. CaVβ2a blocks inhibition unmasking latent enhancement. Moreover, using mutants and chimeras I found palmitoylation of CaVβ2a at the N-terminus plays a key role in blocking inhibition. My findings predict the expression and localization of different CaVβ isoforms would dramatically alter modulation of N-current and thus may represent a previously unrecognized form of plasticity.
The inhibition of N-current by M1Rs is controversial. It has been proposed recently that inhibition is directly attributable to the depletion of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during M1 stimulation. However, in our lab, we have found arachidonic acid (AA) release, which occurs subsequent to PtdIns(4,5)P2 hydrolysis, is both necessary and sufficient to elicit inhibition. Therefore, in a second study, I tested the effect of CaVβ expression on N-current during exogenous AA application and found a pattern of modulation identical to M1R stimulation. Furthermore, I took part in a collaborative project identifying the AA producing enzyme, diacylglycerol lipase (DAGL), to be a necessary component of the inhibitory pathway elicited by M1Rs. These findings provide increased evidence for AA release being a key factor in the M1R stimulated pathway of inhibition. Moreover, these discoveries identify the expression of CaVβ2a and use of specific DAGL inhibitors as a molecular and pharmacological strategy to block inhibition of N-current, respectively. These tools allow the dissection of downstream effects of M1R stimulation, so that other modulatory effects may be observed.
The phosphorylation of N-channels by protein kinase C (PKC) blocks inhibition of current brought on by G-protein β and γ subunits (Gβγ) binding directly to the channel. Relief of Gβγ inhibition by other means has been identified as a mechanism of short term plasticity. M1Rs are known to simulate PKC, but a connection between M1Rs and PKC phosphorylation of Nchannels had not been demonstrated. I hypothesized that PKC stimulation may be occluded by other downstream effects of M1Rs. Therefore in a third study, I used a pharmacological approach on SCG neurons to dissect the PKC activating pathway from the other downstream effects of M1 stimulation. I observed modulation of N-current indicating a loss of Gβγ&#; inhibition, thus consistent with PKC phosphorylation of channels. This conclusion reveals another aspect of M1 modulation, which can function as a means of short term plasticity.
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Khan, J. "Investigation into the effects of specific muscarinic acetylcholine receptor antagonists on the myocardium in pre-clinical conditions of ischaemia reperfusion injury and oxidative stress model." Thesis, Coventry University, 2015. http://curve.coventry.ac.uk/open/items/3508a32c-8427-4ffe-9134-704a3e8c304b/1.
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Muscarinic acetylcholine receptors (mAChRs) are G-protein coupled receptors that mediate various actions of Acetylcholine (ACh) in the central nervous system and peripheral nervous system. In mammals, five distinct mAChR subtypes (M1-M5) have been recognised with the M2 subtype being predominantly present in the heart. The mAChR antagonists are routinely used for the treatment of various pathophysiological conditions including respiratory conditions. However, it has been postulated that mAChR antagonists may increase morbidity and mortality in chronic obstructive pulmonary disorder (COPD) and asthma patients with underlying cardiovascular disease, raising concerns regarding the cardiovascular safety of these agents. The current study was therefore undertaken to investigate the effects of individual mAChR antagonists in the setting of myocardial ischaemia reperfusion injury and oxidative stress models. We also investigated whether the inhibition of the mitochondrial permeability transition pore (MPTP) with cyclosporine-A (CsA) in the presence and absence of individual mAChR antagonists provided protection against ischaemia reperfusion injury. Furthermore, we also aimed to investigate the intracellular signalling pathway associated with mAChRs antagonists mediated myocardial injury under the stress conditions. Langendorff results showed that the non-selective M1-M3 mAChR antagonist, ipratropium bromide, the M2 mAChR antagonist, AF-DX 116 and the M3 mAChR antagonist, DAU 5884 significantly increased the infarct size to risk ratio of the heart in conditions of ischaemia and reperfusion. Detrimental effects of AF-DX 116 and DAU 5884 were abrogated by co-treatment of these drugs with mAChR agonist, acetylcholine (ACh) and/or CsA. Cell viability data of isolated cardiac myocytes revealed that AF-DX 116 and DAU 5884 caused a concentration dependent decrease in the viability of cardiac myocytes as well as causing a reduction in the time taken to depolarisation and hypercontracture under oxidative stress. AF-DX 116 and DAU 5884 significantly increased the levels of p-SAPK/JNK and decreased the levels of p-Akt and p-ERK. In addition, ACh and CsA showed to activate p-Akt and p-ERK. To conclude, the data suggest that AF-DX 116 and DAU 5884 caused cardiotoxicity at cellular, tissue and protein level in conditions of ischaemia reperfusion injury and oxidative stress. Furthermore, inhibition of the mitochondrial transition pore with CsA protected against the AF-DX 116 and DAU 5884 induced injury via activation of the pro-survival proteins, p-Akt and p-ERK.
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Khoza, Kenneth. "Characterisation of the α2A-adrenoceptor antagonism by mirtazapine and its modifying effects on receptor signalling / Kenneth Khoza." Thesis, North-West University, 2004. http://hdl.handle.net/10394/673.
Full textAbstract:
Mirtazapine is an atypical antidepressant employed clinically for the treatment of major
depression. As a multipotent antagonist it acts at α2a-adrenergic receptors (α2a -ARs).
serotonin type-2A receptors (5-HT2a-Rs) and histamine type-I receptors (H1-Rs). Its actions
at the α2a-AR have been proposed to play a role in its putative earlier onset of action.
However, it is not known whether mirtazapine is a neutral antagonist or inverse agonist at α2a-
ARs. The current study aimed to determine the mode of α2a-AR antagonism by mirtazapine,
as well as to investigate in vitro the modulatory effects of mirtazapine pre-treatments on β-adrenergic
receptor (β-AR), muscarinic acetylcholine receptor (mAChR) and α2a-AR
functions.
Chinese hamster ovary (CHO-K1) cells expressing the porcine α2a-AR at high numbers (α2a-H),
a constitutively active mutant α2a-AR (α2a--CAM), or mock-transfected control cells (neo)
were radio-labelled with [3H]-adenine and concentration-effect curves of mirtazapine,
yohimbine, mianserin or idazoxan were constructed, measuring [3H]-cAMP accumulation. In
addition human neuroblastoma SH-SY5Y cells and CHO-K1 cells expressing the porcine α2a-
AR at low numbers (am-L) were used to investigate the effect of mirtazapine pre-treatments
on mAChRs and β-ARS or α2a-ARs respectively. After radio-labelling with myo-[2-3H]-inositol
or [2-%]-adenine, radio-label uptake was measured and receptor function was investigated
by constructing concentration-effect curves, measuring [3H]-IPx or [3H]-cAMP accumulation
respectively.
The results from the current study show that mirtazapine binds to the α2a-AR with an affinity
value in the lower micromolar range (K1≈ 0.32 µM; pK1 = 6.50 ± 0.07). Mirtazapine is not a
partial agonist at α2a-ARs as it does not affect [3H]-cAMP accumulation in α2a-H cells.
Preliminary results suggest that mirtazapine displays partial inverse agonism in α2a-CAM
cells, while mianserin displays neutral antagonism. Mirtazapine pre-treatment in SH-SY5Y
cells does not alter muscarinic receptor function (different from fluoxetine and imipramine),
but reduces I-isoproterenol-induced increase in [3H]-cAMP accumulation in SH-SY5Y cells
(typically associated with chronic antidepressant activity). Although inconclusive, the data
also suggests that mirtazapine may reduce α2a-AR function.Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.
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Eager, Blenerhassit Edward. "The modulating effect of sildenafil on cell viability and on the function of selected pharmacological receptors in cell cultures / B.E. Eagar." Thesis, North-West University, 2004. http://hdl.handle.net/10394/611.
Full textAbstract:
Since sildenafil's (Viagra®), a phospodiesterase type 5 (PDE5) inhibitor, approval for the
treatment of male erectile dysfunction (MED) in the United States early 1998, 274
adverse event reports were filed by the Food and Drug Administration (FDA) between 4
Jan. 1998 and 21 Feb. 2001 with sildenafil as the primary suspect of various
neurological disturbances, including amnesia and aggressive behaviour (Milman and
Arnold, 2002). These and other research findings have prompted investigations into the
possible central effects of sildenafil.
The G protein-coupled muscarinic adetylcholine receptors (mAChRs) and serotonergic
receptors (5HT-Rs), have been linked to antidepressant action (Brink et al. 2004).
GPCRs signal through the phosphatidylinositol signal transduction pathway known to
activate protein kinases (PKs). Since the nitric oxide (NO)-guanylyl cyclase signal
transduction pathway is also known to involve the activation of PKs (via cyclic guanosine
monophosphate (cGMP)), the scope is opened for sildenafil to possibly modulate the
action of antidepressants by elevating cGMP levels.
It is generally assumed that excitotoxic delayed cell death is pathologically linked to an
increase in the release of excitatory neurotransmitters e.g. glutamate. Glutamate
antagonists, especially those that block the define NMDA-receptors, are neuroprotective,
showing the importance of the NMDA-NO-cGMP pathway in neuroprotection (Brandt et
al., 2003). Sildenafil may play a role in neuroprotection by elevating cGMP levels.
Aims: The aims of the study were to investigate any neuroprotective properties of
sildenafil, as well as modulating effects of sildenafil pre-treatment on mAChR function.
Methods: Human neuroblastoma SH-SY5Y or human epithelial HeLa cells were seeded
in 24-well plates and pre-treated for 24 hours in serum-free medium with no drug
(control), PDE5 inhibitors sildenafil (100nM and 450 nM), dipiridamole (20 µM) or
zaprinast (20 µM), non-selective PDE inhibitor 3-isobutyl-I-methylxanthine (IBMX -
ImM), cGMP analogue N2,2'-0-dibutyrylguanosine 3'5'-cyclic monophosphate sodium
salt (500 µM), guanylcyclase inhibitor 1H-[1 ,2,4]oxadiazolo[4,3-a]quinoxalin-I-one (ODQ
- 3 µM) or sildenafil + ODQ (450 nM and 3 µM respectively). Thereafter cells were used
to determine mAChR function by constructing dose-response curves of methacholine or
to determine cell viability utilising the Trypan blue, propidium iodide and MTT tests for
cell viability.
Results: Sildenafil pre-treatments induced a 2.5-fold increase in ,the Emax value of
methacholine in neuronal cells but did not show a significant increase in epithelial cells
The Trypan blue test suggests that neither the PDE5 inhibitors nor a cGMP analogue
show any neuroprotection. Rather, sildenafil 450 nM, dipiridamole and IBMX displayed
a neurodegenerative effect. The MTT test was not suitable, since pre-treatment with the
abovementioned drugs inhibited the formation of forrnazan. The propidium iodide assay
could also not be used, due to severe cell loss.
Conclusion: Sildenafil upregulates mAChR function in SH-SY5Y cells and displays a
neurodegenerative, and not a protective property, in neuronal cells. This is not likely to
be associated with its PDE5 inhibitory action, but may possibly be linked to an increase
in cGMP levels via the NO-cGMP pathway.Thesis (M.Sc. (Pharmacology))--North-West University, Potchefstroom Campus, 2005.
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Pontes, Carolina Nobre Ribeiro. "Influência da Angiotensina-(1-7) na sensibilidade colinérgica cardíaca de ratos normotensos e hipertensos." Universidade Federal de Goiás, 2018. http://repositorio.bc.ufg.br/tede/handle/tede/8940.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Previous studies suggested that the Angiotensin-(1-7) [(Ang-(1-7)] is able to modulate the cardiac sympathetic control and beta-adrenergic sensitivity. However, whether or not Ang-(1- 7) modulates the cholinergic activity in the heart remains unknown. The aim of this study was to evaluate the influence of Ang-(1-7) upon cholinergic sensitivity of hearts from normotensive and hypertensive rats. Wistar and Spontaneously Hypertensive Rats (SHR) were anesthetized with urethane and underwent catheterization of femoral artery and left ventricle to record the arterial and intraventricular pressure, respectively. Following, a dose-response curve of acetylcholine (ACh, 10, 20, 40 and 80 ng/Kg, i.v. into femoral vein) was performed in the absence or presence of Ang-(1-7) (7 x 10-12 mol/min), Mas receptor antagonist A-779 (7 x 10-11 mol/min) or Ang-(1-7)+A-779. Isolated hearts were perfused according to the Langendorff technique. Increasing concentrations of ACh (10-7 to 10-5 mol/L) were added to the hearts in absence or presence of Ang-(1-7), (2 x 10-11 mol/L), A-779, (2 x 10-10 mol/L), Ang-(1-7)+A-779, MrgD receptor antagonist, D-PRO (2 x 10-10 mol/L) or D-PRO+Ang-(1-7). ACh-induced vasorelaxation was assessed in absence or presence of Ang-(1-7) (2 x 10-11 mol/L or 2 x 10-10 mol/L). Ang-(1-7) attenuated the effect of ACh in decreasing the intraventricular systolic, dP/dt max and dP/dt min in anesthetized Wistar and SHR. These effects were blocked by A-779. Ang-(1-7) did not change the amplitude of the hypotensive effect evoked by ACh in Wistar or SHRs. In isolated hearts, Ang-(1-7) also attenuated the reduction of the intraventricular systolic pressure, dP/dt max and dP/dt min evoked by ACh. A-779 blocked the Ang-(1-7) effects in hearts from Wistar. A-779 or D-PRO did not modify the effects of Ang-(1-7) in hearts from SHR, but in presence of D-PRO, Ang-(1-7) effects were equipotent. Ang-(1-7) attenuated the vasorelaxation induced by ACh in aorta from SHR by only in SHR group. These data suggest that Ang-(1-7) exerts differential modulation of cardiac cholinergic sensitivity during experimental primary hypertension, which is independent on blood pressure.
Estudos prévios sugerem que a Angiotensina-(1-7) [(Ang-(1-7)] é capaz de modular o controle simpático cardíaco e sensibilidade beta-adrenérgica. Entretanto, ainda não se sabe se a Ang-(1-7) consegue modular a atividade colinérgica no coração. O objetivo deste estudo foiavaliar a influência da Ang-(1-7) na sensibilidade colinérgica cardíaca de ratos normotensos e hipertensos. Wistar e Ratos Espontaneamente Hipertensos (SHR) foram anestesiados com uretano e submetidos à canulação de artéria femoral e ventrículo esquerdo cardíaco para registro de pressão arterial e intraventricular, respectivamente. Em seguida, foi realizada uma curva dose-resposta de acetilcolina (ACh, 10, 20, 40 e 80 ng/Kg, i.v.) por infusão pela veia femoral. A infusão ocorreu na presença e ausência de Ang-(1-7) (7 x 10-12 mol/min), do antagonista do receptor Mas, A-779 (7 x 10-11 mol/min) ou de Ang-(1-7)+A-779. Os corações isolados foram perfundidos de acordo com a técnica de Langendorff e concentrações crescentes de ACh (10-7 a 10-5 mol/L) foram adicionadas aos corações na presença ou ausência de Ang-(1-7), (2 x 10-11 mol/L), A-779, (2 x 10-10 mol/L), Ang-(1-7)+A-779, antagonista do receptor MrgD, D-PRO (2 x 10-10 mol/L) ou D-PRO+Ang-(1-7). O vasorrelaxamento induzido pela ACh foi mensurado na presença ou ausência da Ang-(1-7) (2 x 10-11 mol/L ou 2 x 10-10 mol/L). Em Wistar e SHR anestesiados, a Ang-(1-7) atenuou o efeito da ACh na queda da pressão intraventricular sistólica, dP/dt máx, e dP/dt mín. Estes efeitos foram bloqueados pelo A-779. A Ang-(1-7) não alterou a resposta hipotensora da ACh em Wistar ou SHR. Nos corações isolados, a Ang-(1-7) também atenuou a redução na pressão intraventricular sistólica, dP/dt máx e dP/dt mín evocados pela ACh. O A-779 bloqueou os efeitos da Ang-(1-7) em corações de Wistar. O A-779 ou D-PRO, per se, não modificaram os efeitos da Ang-(1-7) em corações de SHR, mas na presença do D-PRO, a Ang-(1-7) apresentou efeitos similares. O vasorrelaxamento da aorta induzido pela ACh foi atenuado pela Ang-(1-7) apenas nos SHR. Estes dados sugerem que a Ang-¬(1-¬7) modula o sistema colinérgico cardíaco de forma diferente no modelo de hipertensão primária experimental e de maneira independente de ajustes na pressão arterial.
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Obied, Taghrid Y. "Prediction of In-Vivo Antimuscarinic Activity (AMA) by In-Vitro Receptor Binding Assessment and PK/PD Modeling For Prototypical Drugs." VCU Scholars Compass, 2007. http://scholarscompass.vcu.edu/etd/1348.
Full textAbstract:
Purpose: To establish a tool, termed as antimuscarinic activity (AMA), to predict the incidence of antimuscarinic adverse events (AMAEs).Methods: A literature review, focused on drugs having off-target interaction with muscarinic receptors, was performed. Prototypical drugs olanzapine, diphenhydramine, paroxetine were selected for the analysis. Scopolamine and darifenacin were included as positive and negative controls, respectively. Physiochemical properties, pharmacokinetic data, and clinical incidence of AMAEs for the selected drugs were collected from reported literature. Extrapolation of literature data was carried-out to obtain exposure data. To determine the drugs muscarinic affinity (Ki values), experiments were performed using 3H-QNB and membrane suspensions of M1, M2, and M3. Cmax, values were combined with Ki values to generate the relevant AMA. Validation of the AMA biomarker was carried-out against the reported AMAEs incidence. Results: With the exclusion of scopolamine and olanzapine for CNS and peripheral AMAEs, respectively, AMA ranking was related to the drugs AMAEs.
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Tribut, Florence. "Effets de l'axotomie sur les propriétés électrophysiologiques et pharmacologiques des récepteurs cholinergiques des cellules neurosécrétrices : les dorsal unpaired median (DUM) neurones du dernier ganglion abdominal de la blatte periplaneta americana L." Angers, 1994. http://www.theses.fr/1994ANGE0011.
Full textAbstract:
Une étude des effets d'une axotomie sur certaines propriétés électrophysiologiques et pharmacologiques d'un type particulier de cellules neurosécrétrices adultes, les dorsal unpaired median (DUM) neurones du dernier ganglion abdominal de la blatte p. Americana a été réalisée. Une étude comparative de l'activité électrique des DUM neurones in situ et isoles à l'aide des techniques de microélectrode intracellulaire et de patch-clamp (configuration cellule entière), a montré que l'augmentation significative de l'amplitude des potentiels d'action, observée des 48h après axotomie, était due à l'augmentation de l'amplitude du courant entrant sodique responsable de la phase de dépolarisation. L'utilisation d'inhibiteurs de la synthèse protéique a permis de démontrer qu'une néosynthèse de canaux sodiques était impliquée dans cette augmentation. Une étude similaire a été réalisée sur les propriétés pharmacologiques des récepteurs cholinergiques de ces cellules. Deux types distincts de récepteurs cholinergiques (un récepteur nicotinique résistant à l'alphabungarotoxine et un récepteur muscarinique de sous type m1 like) ont été caractérisés pharmacologiquement sur les DUM neurones in situ. L'axotomie produit une rapide diminution de la sensibilité de ces deux types de récepteurs vis-à-vis de cet agoniste. De plus, un troisième récepteur a été caractérisé et identifié comme étant un récepteur mixte (nicotinique/muscarinique). Tous les effecteurs contrôlés par ces différents types de récepteurs ont été mis en évidence.
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Roberts-Crowley, Mandy L. "Modulation of Cav1.3 L-Type Calcium Channels by Arachidonic Acid and Muscarinic M1 Receptors: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/348.
Full textAbstract:
Membrane excitability, gene expression, and neurotransmitter release are all controlled by voltage-gated L-type Ca2+ (L- )channels. In turn, Ca2+ channels are highly regulated by signal transduction cascades initiated by G protein-coupled receptor (GPCR) activation. In medium spiny neurons of the striatum, both the muscarinic M1 receptors (M1R) and dopaminergic D2 receptors (D2R) specifically inhibit the Cav1.3 L-channel.
In Chapters III and IV, the pathways downstream of M1Rs and D2Rs are examined to determine whether an overlap or intersection in inhibition of Cav1.3 occurs by these two receptors. Transient transfection of Cav1.3 channels in HEK 293 cells, stably transfected with the M1R, and in ST14A cells were used as model systems. While a further characterization of ST14A cells determined that they exhibit a striatal profile, D2Rs or M1Rs did not inhibit Cav1.3. Lack of current inhibition may be due to the finding of no detectable expression of phospholipase Cβ-1 protein in ST14A cells.
Ca2+ channels are multiprotein complexes comprised of α1, β, and α2δ subunits. While the actions of arachidonic acid (AA) have been shown to mimic M1R inhibition of L-current in superior cervical ganglion neurons, the precise identity of the L-channel in these neurons -either Cav1.2 or Cav1.3 or both- is not known. The transfected model systems allowed for the analysis of whole-cells currents with different β subunit combinations as well as the study of only Cav1.3 channels. In Chapter III, I show that activation of M1Rs with the agonist Oxo-M inhibited Cav1.3 channels coexpressed with either β1b, β2a, β3, or β4 subunits. Surprisingly, the magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits. In Chapter V, AA is shown to mimic the profile of M1R stimulation on Cav1.3 currents. The magnitude of Cav1.3, β2a currents was inhibited less than Cav1.3 currents with other β subunits by AA. This discovery points to a novel role for accessory β subunits in altering the magnitude of AA inhibition and kinetic changes of Cav1.3.
Arachidonic acid (AA) inhibits Ca2+ channels by an unknown mechanism at an unknown site. In Chapter V, I found that Cavl.3 inhibition by AA was state-dependent and most likely stabilizes a closed channel conformation. The finding that the Ca2+ channel accessory β subunit alters the magnitude of AA inhibition and kinetic changes of Cav1.3 suggests that AA could alter processes which rely on L-channels such as Ca2+-dependent gene expression, secretion and membrane excitability.
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Warrier, Sunita. "cAMP BIOSENSORS AND SPATIOTEMPORAL cAMP SIGNALING IN ADULT CARDIAC MYOCYTES." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1175718415.
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Greverath, Lena Maria [Verfasser]. "Untersuchung zur Bedeutung genetischer Polymorphismen des muskarinergen Acetylcholinrezeptors Typ 3 bei chronisch-entzündlichen Gallengangserkrankungen : Investigation of Muscarinic Acetylcholine Receptor Type 3 Gene Polymorphisms in chronic inflammatory bile duct diseases / Lena Maria Greverath." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2021. http://d-nb.info/1228860661/34.
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Sihver, Sven. "Development of in vitro and ex vivo positron-emitting tracer techniques and their application to neurotrauma." Doctoral thesis, Uppsala universitet, Institutionen för neurovetenskap, 2000. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-485.
Full textAbstract:
The use of positron-emitting tracers has been extended beyond tomographic facilities in the last few years, giving rise to a general positron-emitting tracing technique. The methodological part of the present thesis involved the evaluation of the performance of storage phosphor (SP) plates, with tracers labeled with high-energy, short-lived, positron-emitting radionuclides, using homogenized tissue specimens and autoradiography with frozen brain sections. The SP plates showed superior sensitivity and a linear response over a wide radioactivity range. Autoradioradiography provided reliable results due to (a) adequate sensitivity for low radioactivity concentration, b) an excellent linear range, and (c) satisfactory resolution. Though equilibration time of receptor-ligand interaction was dependent upon section thickness, quantification was possib with thinner sections. An initial finding using frozen section autoradiography of rat brain and spinal cord showed preferential binding of [11C]4-NMPB, a muscarinic acetylcholine (mACh) receptor antagonist, to the M4 subtype of mACh receptors. Further work to ascertain this specificity, by use of binding studies on cell membranes from CHO-K1 cells expressing individual subtypes of human mACh receptors, suggested lack of subtype selectivity. With respect to the possible cliinical use in glutamatergic neuropathology, [11C]cyano-dizocilpine, as a potential PET tracer for the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, was studied. The in vivo visualization of specific binding could not be achieved, though in vitro binding demonstrated good specificity and preferential binding to the activated for of the NMDA receptors. The use of the glucose analogue [18F]fluorodeoxyglucose (FDG) to study glucose utilization was evaluated in experimental traumatic brain injury (TBI). A trauma-induced increased uptake of FDG was seen, whereas the uptake of [1-14C]glucose remained unchanged. This discrepancy might be due to the increased postraumatic affinity of FDG for the endothelial glucose transporter proteins and/or to the hexokinase enzyme. [11C]Cyano-dizocilpine, [11C]4-NMPB, and [11C]flumazenil were utilized in autoradiography to evaluate changes in NMDA, mACh, and GABAA receptors, espectively, in experimental TBI. Observations showed a global decrease in the binding potential BP) of (i) [11C]cyano-dizocilpine acutely and 12 hrs after TBI, and (ii) of [11C]4-NMPB at 12 hrs after TBI, and (iii) a decrease in the BP of [11C]flumazenil in the cortex and hippocampus ipsilateral to the site of injury. The demonstrated changes in receptor binding after TBI are indicative of a widely dissipated effect of TBI on the particular neurotransmitter receptor systems as compared with what would be expected from FDG studies after TBI, i.e., a local disturbed neurotransmission.
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Bodenstein, Johannes. "Antagonism by selected classical irreversible competitive antagonists : an investigation into the proposed non-specific mechanisms involved / Johannes Bodenstein." Thesis, North-West University, 2003. http://hdl.handle.net/10394/92.
Full textAbstract:
Many irreversible antagonists are known to bind irreversibly to pharmacological
receptors. However, few studies suggest that these irreversible antagonists may also
display irreversible non-specific antagonism by binding irreversibly to non-syntopic
binding sites on the receptor macromolecule, whereby they modulate the signal
transduction of these receptors or reduce the agonist binding affmity.
The aim of this study was to investigate whether the classical irreversible antagonists
phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific
antagonism at various G protein-coupled receptor (GPCR) types. In addition,
the subcellular mechanism whereby benextramine displays irreversible non-specific
antagonism was investigated.
Three cell lines were employed to investigate the antagonism by these irreversible
antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine
a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human
neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine
receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express
the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were
pre-treated at the appropriate concentrations and incubation times with an appropriate
irreversible antagonist, with or without an appropriate reversible competitive antagonist
at a sufficient concentration to protect the specific receptors. This was followed by
washing procedures with drug-free media to rinse any unbound or reversibly bound
drugs from the cells. When appropriate, cell membranes were prepared. Receptor
function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation,
or the binding of [35S]-GTPyS to membraness. Receptor concentrations were
determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated.
Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10
uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent.
When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by
inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated
effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively.
4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific
antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism
was observed when the incubation time was increased (100 uM, 60 minutes).
In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP
mustard display irreversible non-specific antagonism at typical experimental
conditions. These findings confirm concerns in literature and supports the possibility
that more irreversible antagonists could display irreversible non-specific antagonism,
and that could influence the interpretation of data obtained with such drugs. In
addition, benextramine may prove to be a useful experimental drug in studying GPCR
signalling.Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.
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Klawonn, Anna. "Molecular Mechanisms of Reward and Aversion." Doctoral thesis, Linköpings universitet, Centrum för social och affektiv neurovetenskap, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-143459.
Full textAbstract:
Various molecular pathways in the brain shape our understanding of good and bad, as well as our motivation to seek and avoid such stimuli. This work evolves around how systemic inflammation causes aversion; and why general unpleasant states such as sickness, stress, pain and nausea are encoded by our brain as undesirable; and contrary to these questions, how drugs of abuse can subjugate the motivational neurocircuitry of the brain. A common feature of these various disease states is involvement of the motivational neurocircuitry - from mesolimbic to striatonigral pathways. Having an intact motivational system is what helps us evade negative outcomes and approach natural positive reinforcers, which is essential for our survival. During disease-states the motivational neurocircuitry may be overthrown by the molecular mechanisms that originally were meant to aid us. In study I, to investigate how inflammation is perceived as aversive, we used a behavioral test based on Pavlovian place conditioning with the aversive inflammatory stimulus E. coli lipopolysaccharide (LPS). Using a combination of cell-type specific gene deletions, pharmacology, and chemogenetics, we uncovered that systemic inflammation triggered aversion by MyD88-dependent activation of the brain endothelium followed by COX1-mediated cerebral prostaglandin E2 (PGE2) synthesis. Moreover, we showed that inflammation-induced PGE2 targeted EP1 receptors on striatal dopamine D1 receptor–expressing neurons and that this signaling sequence induced aversion through GABA-mediated inhibition of dopaminergic cells. Finally, inflammation-induced aversion was not an indirect consequence of fever or anorexia but constituted an independent inflammatory symptom triggered by a unique molecular mechanism. Collectively, these findings demonstrate that PGE2-mediated modulation of the dopaminergic circuitry is a key mechanism underlying inflammation-induced aversion. In study II, we investigate the role of peripheral IFN-γ in LPS induced conditioned place aversion by employing a strategy based on global and cell-type specific gene deletions, combined with measures of gene-expression. LPS induced IFN-ɣ expression in the blood, and deletion of IFN-ɣ or its receptor prevented conditioned place aversion (CPA) to LPS. LPS increased the expression of chemokine Cxcl10 in the striatum of normal mice. This induction was absent in mice lacking IFN-ɣ receptors or Myd88 in blood brain barrier endothelial cells. Furthermore, inflammation-induced aversion was blocked in mice lacking Cxcl10 or its receptor Cxcr3. Finally, mice with a selective deletion of the IFN-ɣ receptor in brain endothelial cells did not develop inflammation-induced aversion. Collectively, these findings demonstrate that circulating IFN-ɣ binding to receptors on brain endothelial cells which induces Cxcl10, is a central link in the signaling chain eliciting inflammation-induced aversion. In study III, we explored the role of melanocortin 4 receptors (MC4Rs) in aversive processing using genetically modified mice in CPA to various stimuli. In normal mice, robust aversions were induced by systemic inflammation, nausea, pain and kappa opioid receptor-induced dysphoria. In sharp contrast, mice lacking MC4Rs displayed preference towards most of the aversive stimuli, but were indifferent to pain. The unusual flip from aversion to reward in mice lacking MC4Rs was dopamine-dependent and associated with a change from decreased to increased activity of the dopamine system. The responses to aversive stimuli were normalized when MC4Rs were re-expressed on dopamine D1 receptor-expressing cells or in the striatum of mice otherwise lacking MC4Rs. Furthermore, activation of arcuate nucleus proopiomelanocortin neurons projecting to the ventral striatum increased the activity of striatal neurons in a MC4R-dependent manner and elicited aversion. Our findings demonstrate that melanocortin signaling through striatal MC4Rs is critical for assigning negative motivational valence to harmful stimuli. The neurotransmitter acetylcholine has been implied in reward learning and drug addiction. However, the role of cholinergic receptor subtypes in such processes remains elusive. In study IV we investigated the function of muscarinic M4Rs on dopamine D1R expressing neurons and acetylcholinergic neurons, using transgenic mice in various reward-enforced behaviors and in a “waiting”-impulsivity test. Mice lacking M4-receptors from D1-receptor expressing neurons exhibited an escalated reward seeking phenotype towards cocaine and natural reward, in Pavlovian conditioning and an operant self-administration task, respectively. In addition, the M4-D1RCre mice showed impaired waiting impulsivity in the 5-choice-serial-reaction-time-task. On the contrary, mice without M4Rs in acetylcholinergic neurons were unable to learn positive reinforcement to natural reward and cocaine, in an operant runway paradigm and in Pavlovian conditioning. Immediate early gene expression mirrored the behavioral findings arising from M4R-D1R knockout, as cocaine induced cFos and FosB was significantly increased in the forebrain of M4-D1RCre mice, whereas it remained normal in the M4R-ChatCre mice. Our study illustrates that muscarinic M4Rs on specific neural populations, either cholinergic or D1R-expressing, are pivotal for learning processes related to both natural reward and drugs of abuse, with opposing functionality.