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Liu, Hongtao, Josefa Hofmann, Inbar Fish, Benjamin Schaake, Katrin Eitel, Amelie Bartuschat, Jonas Kaindl, et al. "Structure-guided development of selective M3 muscarinic acetylcholine receptor antagonists." Proceedings of the National Academy of Sciences 115, no. 47 (November 7, 2018): 12046–50. http://dx.doi.org/10.1073/pnas.1813988115.
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Drugs that treat chronic obstructive pulmonary disease by antagonizing the M3 muscarinic acetylcholine receptor (M3R) have had a significant effect on health, but can suffer from their lack of selectivity against the M2R subtype, which modulates heart rate. Beginning with the crystal structures of M2R and M3R, we exploited a single amino acid difference in their orthosteric binding pockets using molecular docking and structure-based design. The resulting M3R antagonists had up to 100-fold selectivity over M2R in affinity and over 1,000-fold selectivity in vivo. The crystal structure of the M3R-selective antagonist in complex with M3R corresponded closely to the docking-predicted geometry, providing a template for further optimization.
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Xie, Guofeng, Cinthia Drachenberg, Masahisa Yamada, Jürgen Wess, and Jean-Pierre Raufman. "Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors." American Journal of Physiology-Gastrointestinal and Liver Physiology 289, no. 3 (September 2005): G521—G529. http://dx.doi.org/10.1152/ajpgi.00105.2004.
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Muscarinic cholinergic mechanisms play a key role in stimulating gastric pepsinogen secretion. Studies using antagonists suggested that the M3 receptor subtype (M3R) plays a prominent role in mediating pepsinogen secretion, but in situ hybridization indicated expression of M1 receptor (M1R) in rat chief cells. We used mice that were deficient in either the M1 (M1R−/−) or M3 (M3R−/−) receptor or that lacked both receptors (M1/3R−/−) to determine the role of M1R and M3R in mediating cholinergic agonist-induced pepsinogen secretion. Pepsinogen secretion from murine gastric glands was determined by adapting methods used for rabbit and rat stomach. In wild-type (WT) mice, maximal concentrations of carbachol and CCK caused a 3.0- and 2.5-fold increase in pepsinogen secretion, respectively. Maximal carbachol-induced secretion from M1R−/− mouse gastric glands was decreased by 25%. In contrast, there was only a slight decrease in carbachol potency and no change in efficacy when comparing M3R−/− with WT glands. To explore the possibility that both M1R and M3R are involved in carbachol-mediated pepsinogen secretion, we examined secretion from glands prepared from M1/3R−/− double-knockout mice. Strikingly, carbachol-induced pepsinogen secretion was nearly abolished in glands from M1/3R−/− mice, whereas CCK-induced secretion was not altered. In situ hybridization for murine M1R and M3R mRNA in gastric mucosa from WT mice revealed abundant signals for both receptor subtypes in the cytoplasm of chief cells. These data clearly indicate that, in gastric chief cells, a mixture of M1 and M3 receptors mediates cholinergic stimulation of pepsinogen secretion and that no other muscarinic receptor subtypes are involved in this activity. The development of a murine secretory model facilitates use of transgenic mice to investigate the regulation of pepsinogen secretion.
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Tuomi, Jari M., Peter Chidiac, and Douglas L. Jones. "Evidence for enhanced M3 muscarinic receptor function and sensitivity to atrial arrhythmia in the RGS2-deficient mouse." American Journal of Physiology-Heart and Circulatory Physiology 298, no. 2 (February 2010): H554—H561. http://dx.doi.org/10.1152/ajpheart.00779.2009.
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Atrial fibrillation (AF) is the most common arrhythmia seen in general practice. Muscarinic ACh receptors (M2R, M3R) are involved in vagally induced AF. M2R and M3R activate the heterotrimeric G proteins, Gi and Gq, respectively, by promoting GTP binding, and these in turn activate distinct K+ channels. Signaling is terminated by GTP hydrolysis, a process accelerated by regulator of G protein signaling (RGS) proteins. RGS2 is selective for Gq and thus may regulate atrial M3R signaling. We hypothesized that knockout of RGS2 (RGS2−/−) would render the atria more susceptible to electrically induced AF. One-month-old male RGS2−/− and C57BL/6 wild-type (WT) mice were instrumented for intracardiac electrophysiology. Atrial effective refractory periods (AERPs) were also determined in the absence and presence of carbachol, atropine, and/or the selective M3R antagonist darifenacin. Susceptibility to electrically induced AF used burst pacing and programmed electrical stimulation with one extrastimulus. Real-time RT-PCR measured atrial and ventricular content of RGS2, RGS4, M2R, M3R, and M4R mRNA. AERP was lower in RGS2−/− compared with WT mice in both the high right atrium (HRA) (30 ± 1 vs. 34 ± 1 ms, P < 0.05) and mid right atrium (MRA) (21 ± 1 vs. 24 ± 1 ms, P < 0.05). Darifenacin eliminated this difference (HRA: 37 ± 2 vs. 39 ± 2 ms, and MRA: 30 ± 2 vs. 30 ± 1, P > 0.4). RGS2−/− were more susceptible than WT mice to atrial tachycardia/fibrillation (AT/F) induction (11/22 vs. 1/25, respectively, P < 0.05). Muscarinic receptor expression did not differ between strains, whereas M2R expression was 70-fold higher than M3R ( P < 0.01). These results suggest that RGS2 is an important cholinergic regulator in the atrium and that RGS2−/− mice have enhanced susceptibility to AT/F via enhanced M3 muscarinic receptor activity.
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Rossi, Mario, Inigo Ruiz de Azua, Luiz F. Barella, Wataru Sakamoto, Lu Zhu, Yinghong Cui, Huiyan Lu, et al. "CK2 acts as a potent negative regulator of receptor-mediated insulin release in vitro and in vivo." Proceedings of the National Academy of Sciences 112, no. 49 (November 23, 2015): E6818—E6824. http://dx.doi.org/10.1073/pnas.1519430112.
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G protein-coupled receptors (GPCRs) regulate virtually all physiological functions including the release of insulin from pancreatic β-cells. β-Cell M3 muscarinic receptors (M3Rs) are known to play an essential role in facilitating insulin release and maintaining proper whole-body glucose homeostasis. As is the case with other GPCRs, M3R activity is regulated by phosphorylation by various kinases, including GPCR kinases and casein kinase 2 (CK2). At present, it remains unknown which of these various kinases are physiologically relevant for the regulation of β-cell activity. In the present study, we demonstrate that inhibition of CK2 in pancreatic β-cells, knockdown of CK2α expression, or genetic deletion of CK2α in β-cells of mutant mice selectively augmented M3R-stimulated insulin release in vitro and in vivo. In vitro studies showed that this effect was associated with an M3R-mediated increase in intracellular calcium levels. Treatment of mouse pancreatic islets with CX4945, a highly selective CK2 inhibitor, greatly reduced agonist-induced phosphorylation of β-cell M3Rs, indicative of CK2-mediated M3R phosphorylation. We also showed that inhibition of CK2 greatly enhanced M3R-stimulated insulin secretion in human islets. Finally, CX4945 treatment protected mice against diet-induced hyperglycemia and glucose intolerance in an M3R-dependent fashion. Our data demonstrate, for the first time to our knowledge, the physiological relevance of CK2 phosphorylation of a GPCR and suggest the novel concept that kinases acting on β-cell GPCRs may represent novel therapeutic targets.
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Haberberger, Rainer, Reas Scholz, Wolfgang Kummer, and Michaela Kress. "M2-Receptor Subtype Does Not Mediate Muscarine-Induced Increases in [Ca2+]i in Nociceptive Neurons of Rat Dorsal Root Ganglia." Journal of Neurophysiology 84, no. 4 (October 1, 2000): 1934–41. http://dx.doi.org/10.1152/jn.2000.84.4.1934.
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Multiple muscarinic receptor subtypes are present on sensory neurons that may be involved in the modulation of nociception. In this study we focused on the presence of the muscarinic receptor subtypes, M2 and M3 (M2R, M3R), in adult rat lumbar dorsal root ganglia (DRG) at the functional ([Ca2+]i measurement), transcriptional (RT-PCR), and translational level (immunohistochemistry). After 1 day in culture exposure of dissociated medium-sized neurons (20–35 μm diam) to muscarine was followed by rises in [Ca2+]i in 76% of the neurons. The [Ca2+]i increase was absent after removal of extracellular calcium and did not desensitize after repetitive application of the agonist. This rise in [Ca2+]i may be explained by the expression of M3R, which can induce release of calcium from internal stores via inositoltrisphospate. Indeed the effect was antagonized by the muscarinic receptor antagonist atropine as well as by the M3R antagonist, 4-diphenylacetoxy-N-(2 chloroethyl)-piperidine hydrochloride (4-DAMP). The pharmacological identification of M3R was corroborated by RT-PCR of total RNA and single-cell RT-PCR, which revealed the presence of mRNA for M3R in lumbar DRG and in single sensory neurons. In addition, RT-PCR also revealed the expression of M2R, which did not seem to contribute to the calcium changes since it was not prevented by the M2 receptor antagonist, gallamine. Immunohistochemistry demonstrated the presence of M2R and M3R in medium-sized lumbar DRG neurons that also coexpressed binding sites for the lectin I-B4, a marker for mainly cutaneous nociceptors. The occurrence of muscarinic receptors in putative nociceptive I-B4-positive neurons suggests the involvement of these acetylcholine receptors in the modulation of processing of nociceptive stimuli.
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Maeda, Shoji, Qianhui Qu, Michael J. Robertson, Georgios Skiniotis, and Brian K. Kobilka. "Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes." Science 364, no. 6440 (May 9, 2019): 552–57. http://dx.doi.org/10.1126/science.aaw5188.
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Muscarinic acetylcholine receptors are G protein–coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/11 family, whereas M2R and M4R couple to the Gi/o family. Here, we present and compare the cryo–electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA. The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed analysis of these structures provides a framework for understanding the molecular determinants of G-protein coupling selectivity.
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Nakajima, Kenichiro, Shalini Jain, Inigo Ruiz de Azua, Sara M. McMillin, Mario Rossi, and Jürgen Wess. "Minireview: Novel Aspects of M3 Muscarinic Receptor Signaling in Pancreatic β-Cells." Molecular Endocrinology 27, no. 8 (August 1, 2013): 1208–16. http://dx.doi.org/10.1210/me.2013-1084.
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The release of insulin from pancreatic β-cells is regulated by a considerable number of G protein–coupled receptors. During the past several years, we have focused on the physiological importance of β-cell M3 muscarinic acetylcholine receptors (M3Rs). At the molecular level, the M3R selectively activates G proteins of the Gq family. Phenotypic analysis of several M3R mutant mouse models, including a mouse strain that lacks M3Rs only in pancreatic β-cells, indicated that β-cell M3Rs play a key role in maintaining blood glucose levels within a normal range. Additional studies with transgenic M3R mouse models strongly suggest that strategies aimed to enhance signaling through β-cell M3Rs may prove useful in the treatment of type 2 diabetes. More recently, we analyzed transgenic mice that expressed an M3R-based designer receptor in a β-cell–specific fashion, which enabled us to chronically activate a β-cell Gq-coupled receptor by a drug that is otherwise pharmacologically inert. Drug-dependent activation of this designer receptor stimulated the sequential activation of Gq, phospholipase C, ERK1/2, and insulin receptor substrate 2 signaling, thus triggering a series of events that greatly improved β-cell function. Most importantly, chronic stimulation of this pathway protected mice against experimentally induced diabetes and glucose intolerance, induced either by streptozotocin or by the consumption of an energy-rich, high-fat diet. Because β-cells are endowed with numerous receptors that mediate their cellular effects via activation of Gq-type G proteins, these findings provide a rational basis for the development of novel antidiabetic drugs targeting this class of receptors.
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Gautam, Dinesh, Inigo Ruiz de Azua, Jian Hua Li, Jean-Marc Guettier, Thomas Heard, Yinghong Cui, Huiyan Lu, et al. "Beneficial Metabolic Effects Caused by Persistent Activation of β-Cell M3 Muscarinic Acetylcholine Receptors in Transgenic Mice." Endocrinology 151, no. 11 (September 15, 2010): 5185–94. http://dx.doi.org/10.1210/en.2010-0519.
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Previous studies have shown that β-cell M3 muscarinic acetylcholine receptors (M3Rs) play a key role in maintaining blood glucose homeostasis by enhancing glucose-dependent insulin release. In this study, we tested the hypothesis that long-term, persistent activation of β-cell M3Rs can improve glucose tolerance and ameliorate the metabolic deficits associated with the consumption of a high-fat diet. To achieve the selective and persistent activation of β-cell M3Rs in vivo, we generated transgenic mice that expressed the Q490L mutant M3R in their pancreatic β-cells (β-M3-Q490L Tg mice). The Q490L point mutation is known to render the M3R constitutively active. The metabolic phenotypes of the transgenic mice were examined in several in vitro and in vivo metabolic tests. In the presence of 15 mm glucose and the absence of M3R ligands, isolated perifused islets prepared from β-M3-Q490L Tg mice released considerably more insulin than wild-type control islets. This effect could be completely blocked by incubation of the transgenic islets with atropine (10 μm), an inverse muscarinic agonist, indicating that the Q490L mutant M3R exhibited ligand-independent signaling (constitutive activity) in mouse β-cells. In vivo studies showed that β-M3-Q490L Tg mice displayed greatly improved glucose tolerance and increased serum insulin levels as well as resistance to diet-induced glucose intolerance and hyperglycemia. These results suggest that chronic activation of β-cell M3Rs may represent a useful approach to boost insulin output in the long-term treatment of type 2 diabetes.
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Ruffino-Netto, Antonio. "Avaliação do excesso de casos de tuberculose atribuídos a infecção HIV/AIDS: ensaio preliminar." Revista de Saúde Pública 29, no. 4 (August 1995): 279–82. http://dx.doi.org/10.1590/s0034-89101995000400004.
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Propõe-se realizar um exercício de estimação do risco atribuível por cento (RA%) da ocorrência de casos de tuberculose na vigência da co-infecção HIV/AIDS. A seguinte fórmula é apresentada: RA%= p[m2r (hR-h)] + (1-p)[m3r (hR-h)] / p[m1+m2r (hR+1+h)] + (1-p)[m3r (hR+1-h)] x 100 onde: p = proporção infectados pelo BK; r = risco de infecção tuberculosa; h = proporção de infectados pelo HIV; m1 = coeficiente de morbidade reativação endógena; m2 = coeficientes de morbidade de reinfecção exógena; m3 = coeficiente de morbidade tuberculose primária; R = risco relativo de morbidade entre infectados pelo HIV.
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Shinnar, Avraham, David Cunningham, Vijay Saraswat, and Benjamin Herta. "M3R." Proceedings of the VLDB Endowment 5, no. 12 (August 2012): 1736–47. http://dx.doi.org/10.14778/2367502.2367513.
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Han, Mei, Jiamei Lian, Yueqing Su, and Chao Deng. "Cevimeline co-treatment attenuates olanzapine-induced metabolic disorders via modulating hepatic M3 muscarinic receptor: AMPKα signalling pathway in female rats." Journal of Psychopharmacology 36, no. 2 (October 25, 2021): 202–13. http://dx.doi.org/10.1177/02698811211050549.
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Background: Olanzapine is one of the most commonly used antipsychotic drugs; however, its metabolic disorders are the main obstacle in the clinic. Olanzapine is a potent antagonist of the M3 acetylcholine muscarinic receptor (M3R), while the downregulated hepatic M3R-AMPKα signalling pathway is involved in metabolic disorders. Aim: This study investigated the effects of chronic co-treatment with cevimeline (an agonist of M3Rs) in attenuating olanzapine-induced metabolic disorders and the underlying mechanisms. Methods: Forty-eight adult female Sprague-Dawley rats were treated orally with olanzapine (2 mg/kg, 3 times/day (t.i.d.)) and/or cevimeline (9 mg/kg, t.i.d.), or control (vehicle) for 9 weeks. Results: Cevimeline co-treatment significantly attenuated olanzapine-induced body weight gain and glucolipid metabolic disorders. Importantly, cevimeline co-treatment attenuated olanzapine-induced upregulation of M3Rs, while the co-treatment improved olanzapine-induced downregulation of AMPKα in the liver. Cevimeline co-treatment attenuated olanzapine-induced dyslipidaemia by modulating the hepatic M3R-AMPKα downstream pathways. Cevimeline co-treatment also improved lower activated AKT-GSK3β signalling to reverse impairment of glucose metabolism and insulin resistance caused by chronic olanzapine treatment. Conclusion: These results not only support the important role of M3R antagonism and its related AMPKα and downstream pathways in antipsychotic-induced metabolic disorders but also indicate that these pathways might be promising targets for pharmacological intervention to control these side effects caused by antipsychotic therapy.
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Harrington, A. M., C. J. Peck, L. Liu, E. Burcher, J. M. Hutson, and B. R. Southwell. "Localization of muscarinic receptors M1R, M2R and M3R in the human colon." Neurogastroenterology & Motility 22, no. 9 (February 9, 2010): 999—e263. http://dx.doi.org/10.1111/j.1365-2982.2009.01456.x.
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Boos, Johannes, Lino Morris Sawicki, Rotem Shlomo Lanzman, Christoph Thomas, Joel Aissa, Christoph Schleich, Philipp Heusch, Gerald Antoch, and Patric Kröpil. "Metal artifact reduction (MAR) based on two-compartment physical modeling: evaluation in patients with hip implants." Acta Radiologica 58, no. 1 (July 19, 2016): 70–76. http://dx.doi.org/10.1177/0284185116633911.
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Background Artifacts from metallic implants can hinder image interpretation in computed tomography (CT). Image quality can be improved using metal artifact reduction (MAR) techniques. Purpose To evaluate the impact of a MAR algorithm on image quality of CT examinations in comparison to filtered back projection (FBP) in patients with hip prostheses. Material and Methods Twenty-two patients with 25 hip prostheses who underwent clinical abdominopelvic CT on a 64-row CT were included in this retrospective study. Axial images were reconstructed with FBP and five increasing MAR levels (M30–34). Objective artifact strength (OAS) (SIart-SInorm) was assessed by region of interest (ROI) measurements in position of the strongest artifact (SIart) and in an osseous structure without artifact (SInorm) (in Hounsfield units [HU]). Two independent readers evaluated subjective image quality regarding metallic hardware, delineation of bone, adjacent muscle, and pelvic organs on a 5-point scale (1, non-diagnostic; 5, excellent image quality). Artifacts in the near field, far field, and newly induced artifacts due to the MAR technique were analyzed. Results OAS values were: M34: 243.8 ± 155.4 HU; M33: 294.3 ± 197.8 HU; M32: 340.5 ± 210.1 HU; M31: 393.6 ± 225.2 HU; M30: 446.8 ± 224.2 HU and FBP: 528.9 ± 227.7 HU. OAS values were significantly lower for M32–34 compared to FBP ( P < 0.01). For overall subjective image quality, results were: FBP, 2.0 ± 0.2; M30, 2.3 ± 0.8; M31, 2.6 ± 0.5; M32, 3.0 ± 0.6; M33, 3.5 ± 0.6; and M34, 3.8 ± 0.4 ( P < 0.001 for M30–M34 vs. FBP, respectively). Increasing MAR levels resulted in new artifacts in 17% of reconstructions. Conclusion The investigated MAR algorithm led to a significant reduction of artifacts from metallic hip implants. The highest MAR level provided the least severe artifacts and the best overall image quality.
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Jana, Barbara, Jarosław Całka, Katarzyna Palus, and Małgorzata Sikora. "Escherichia coli -induced inflammation changes the expression of acetylcholine receptors (M2R, M3R, and α-7 nAChR) in the pig uterus." Journal of Veterinary Research 64, no. 4 (November 6, 2020): 531–41. http://dx.doi.org/10.2478/jvetres-2020-0073.
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AbstractIntroductionThe influence of inflammation on the patterns of muscarinic 2 and 3 receptor subtypes (M2R and M3R), and α-7 nicotinic acetylcholine receptor (α-7 nAChR) expression in the porcine uterus was investigated.Material and MethodsOn day three of the oestrous cycle of gilts aged 7–8 months with body weight 90–120 kg, either an E. coli suspension (E. coli group, n = 5) or saline (Sal group, n = 5) was administered into the uterine horns via laparotomy or only laparotomy was performed on control swine (Ctrl group, n = 5). After eight days, and the onset of severe acute endometritis in the E. coli group, the uterine mRNA and protein receptor expression levels were determined using real-time RT-PCR and Western blotting, respectively, with receptor localisation by immunofluorescence.ResultsThe studied receptors were in the luminal epithelium, glands, blood vessels, and myometrial muscle cells of all gilts. The M2R mRNA level was lower in the inflamed endometrium compared to the Ctrl and Sal groups. Also in this tissue, the expression of M3R mRNA and protein was lower than in the Ctrl and Sal groups. The M3R protein level in the bacterially challenged myometrium was found to be increased compared to unadministered groups. In the endometrium of the E. coli group, the α-7 nAChR protein level was lower than in the Sal group, and in the myometrium it was reduced in relation to both the other groups. P values were ≤ 0.05 in all cases.ConclusionInflammation causes alterations in the M2R, M3R, and α-7 nAChR expression in the pig uterus, suggesting their significance in the course and repercussions of uterine inflammation.
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Zhu, Lu, Mario Rossi, Amanda Cohen, Jonathan Pham, Hongchao Zheng, Diptadip Dattaroy, Taro Mukaibo, et al. "Allosteric modulation of β-cell M3muscarinic acetylcholine receptors greatly improves glucose homeostasis in lean and obese mice." Proceedings of the National Academy of Sciences 116, no. 37 (August 26, 2019): 18684–90. http://dx.doi.org/10.1073/pnas.1904943116.
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Given the global epidemic in type 2 diabetes, novel antidiabetic drugs with increased efficacy and reduced side effects are urgently needed. Previous work has shown that M3muscarinic acetylcholine (ACh) receptors (M3Rs) expressed by pancreatic β cells play key roles in stimulating insulin secretion and maintaining physiological blood glucose levels. In the present study, we tested the hypothesis that a positive allosteric modulator (PAM) of M3R function can improve glucose homeostasis in mice by promoting insulin release. One major advantage of this approach is that allosteric agents respect the ACh-dependent spatiotemporal control of M3R activity. In this study, we first demonstrated that VU0119498, a drug known to act as a PAM at M3Rs, significantly augmented ACh-induced insulin release from cultured β cells and mouse and human pancreatic islets. This stimulatory effect was absent in islets prepared from mice lacking M3Rs, indicative of the involvement of M3Rs. VU0119498 treatment of wild-type mice caused a significant increase in plasma insulin levels, accompanied by a striking improvement in glucose tolerance. These effects were mediated by β-cell M3Rs, since they were absent in mutant mice selectively lacking M3Rs in β cells. Moreover, acute VU0119498 treatment of obese, glucose-intolerant mice triggered enhanced insulin release and restored normal glucose tolerance. Interestingly, doses of VU0119498 that led to pronounced improvements in glucose homeostasis did not cause any significant side effects due to activation of M3Rs expressed by other peripheral cell types. Taken together, the data from this proof-of-concept study strongly suggest that M3R PAMs may become clinically useful as novel antidiabetic agents.
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Schlenz, Heike, Wolfgang Kummer, Gitte Jositsch, Jürgen Wess, and Gabriela Krasteva. "Muscarinic receptor-mediated bronchoconstriction is coupled to caveolae in murine airways." American Journal of Physiology-Lung Cellular and Molecular Physiology 298, no. 5 (May 2010): L626—L636. http://dx.doi.org/10.1152/ajplung.00261.2009.
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Cholinergic bronchoconstriction is mediated by M2 and M3 muscarinic receptors (MR). In heart and urinary bladder, MR are linked to caveolin-1 or -3, the structural proteins of caveolae. Caveolae are cholesterol-rich, omega-shaped invaginations of the plasma membrane. They provide a scaffold for multiple G protein receptors and membrane-bound enzymes, thereby orchestrating signaling into the cell interior. Hence, we hypothesized that airway MR signaling pathways are coupled to caveolae as well. To address this issue, we determined the distribution of caveolin isoforms and MR subtype M2R in murine and human airways and investigated protein-protein associations by fluorescence resonance energy transfer (FRET)-confocal laser scanning microscopy (CLSM) analysis in immunolabeled murine tissue sections. Bronchoconstrictor responses of murine bronchi were recorded in lung-slice preparations before and after caveolae disruption by methyl-β-cyclodextrin, with efficiency of this treatment being validated by electron microscopy. KCl-induced bronchoconstriction was unaffected after treatment, demonstrating functional integrity of the smooth muscle. Caveolae disruption decreased muscarine-induced bronchoconstriction in wild-type and abolished it in M2R−/− and M3R−/− mice. Thus M2R and M3R signaling pathways require intact caveolae. Furthermore, we identified a presumed skeletal and cardiac myocyte-specific caveolin isoform, caveolin-3, in human and murine bronchial smooth muscle and found it to be associated with M2R in situ. In contrast, M2R was not associated with caveolin-1, despite an in situ association of caveolin-1 and caveolin-3 that was detected. Here, we demonstrated that M2R- and M3R-mediated bronchoconstriction is caveolae-dependent. Since caveolin-3 is directly associated with M2R, we suggest caveolin-3 as novel regulator of M2R-mediated signaling.
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Thor, Doreen, Angela Schulz, Thomas Hermsdorf, and Torsten Schöneberg. "Generation of an agonistic binding site for blockers of the M3 muscarinic acetylcholine receptor." Biochemical Journal 412, no. 1 (April 25, 2008): 103–12. http://dx.doi.org/10.1042/bj20071366.
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GPCRs (G-protein-coupled receptors) exist in a spontaneous equilibrium between active and inactive conformations that are stabilized by agonists and inverse agonists respectively. Because ligand binding of agonists and inverse agonists often occurs in a competitive manner, one can assume an overlap between both binding sites. Only a few studies report mutations in GPCRs that convert receptor blockers into agonists by unknown mechanisms. Taking advantage of a genetically modified yeast strain, we screened libraries of mutant M3Rs {M3 mAChRs [muscarinic ACh (acetylcholine) receptors)]} and identified 13 mutants which could be activated by atropine (EC50 0.3–10 μM), an inverse agonist on wild-type M3R. Many of the mutations sensitizing M3R to atropine activation were located at the junction of intracellular loop 3 and helix 6, a region known to be involved in G-protein coupling. In addition to atropine, the pharmacological switch was found for other M3R blockers such as scopolamine, pirenzepine and oxybutynine. However, atropine functions as an agonist on the mutant M3R only when expressed in yeast, but not in mammalian COS-7 cells, although high-affinity ligand binding was comparable in both expression systems. Interestingly, we found that atropine still blocks carbachol-induced activation of the M3R mutants in the yeast expression system by binding at the high-affinity-binding site (Ki ∼10 nM). Our results indicate that blocker-to-agonist converting mutations enable atropine to function as both agonist and antagonist by interaction with two functionally distinct binding sites.
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Gericke, Adrian, Jan J. Sniatecki, Veronique G. A. Mayer, Evgeny Goloborodko, Andreas Patzak, Jürgen Wess, and Norbert Pfeiffer. "Role of M1, M3, and M5 muscarinic acetylcholine receptors in cholinergic dilation of small arteries studied with gene-targeted mice." American Journal of Physiology-Heart and Circulatory Physiology 300, no. 5 (May 2011): H1602—H1608. http://dx.doi.org/10.1152/ajpheart.00982.2010.
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Acetylcholine regulates perfusion of numerous organs via changes in local blood flow involving muscarinic receptor-induced release of vasorelaxing agents from the endothelium. The purpose of the present study was to determine the role of M1, M3, and M5 muscarinic acetylcholine receptors in vasodilation of small arteries using gene-targeted mice deficient in either of the three receptor subtypes (M1R−/−, M3R−/−, or M5R−/− mice, respectively). Muscarinic receptor gene expression was determined in murine cutaneous, skeletal muscle, and renal interlobar arteries using real-time PCR. Moreover, respective arteries from M1R−/−, M3R−/−, M5R−/−, and wild-type mice were isolated, cannulated with micropipettes, and pressurized. Luminal diameter was measured using video microscopy. mRNA for all five muscarinic receptor subtypes was detected in all three vascular preparations from wild-type mice. However, M3 receptor mRNA was found to be most abundant. Acetylcholine produced dose-dependent dilation in all three vascular preparations from M1R−/−, M5R−/−, and wild-type mice. In contrast, cholinergic dilation was virtually abolished in arteries from M3R−/− mice. Deletion of either M1, M3, or M5 receptor genes did not affect responses to nonmuscarinic vasodilators, such as substance P and nitroprusside. These findings provide the first direct evidence that M3 receptors mediate cholinergic vasodilation in cutaneous, skeletal muscle, and renal interlobar arteries. In contrast, neither M1 nor M5 receptors appear to be involved in cholinergic responses of the three vascular preparations tested.
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Abe, S., H. Tsuboi, F. Honda, H. Takahashi, Y. Kondo, I. Matsumoto, and T. Sumida. "AB0122 DETECTION OF CIRCULATING M3 MUSCARINIC ACETYLCHOLINE RECEPTOR REACTIVE TH17 CELLS IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1361.2–1361. http://dx.doi.org/10.1136/annrheumdis-2020-eular.1642.
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Background:Sjögren’s syndrome (SS) is an autoimmune disease which is characterized by lymphocytic infiltration including CD4+IL-17 producing helper T (Th17) cells to the lacrimal and salivary glands. We previously detected anti-M3 muscarinic acetylcholine receptor (M3R) antibodies (1) and M3R reactive CD4+IFNγ producing helper T (Th1) cells (2) in SS patients. Moreover, we clarified that M3R reactive Th1 and Th17 cells had pathogenic roles in the development of auto-immune sialadenitis in SS mouse model (3).Objectives:The purpose of this study was to identify circulating M3R reactive Th17 cells among primary SS (pSS) patients, and to determine functional properties of those cells.Methods:1)Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of 10 pSS patients, age gender matched 10 healthy controls (HC), and 5 IgG4-related disease (IgG4-RD) patients. According to their HLA-DRB1 typing, top 10 ranked 20 mer peptides from the full length of M3R, which were highly predicted to bind to each HLA molecules according to the immune epitope database website, were selected for each subjects. PBMCs were stimulated with these selected M3R peptides mixed for 40 hours, and M3R peptide reactive IL-17 secreting cells were detected by IL-17 enzyme-linked immunospot assay (ELISpot).2)PBMCs from 5 pSS patients who were positive for M3R specific IL-17 secreting cells, were stimulated with selected 12-20 mer M3R peptides separately, to identify the dominant M3R peptides responsible for IL-17 secretion by ELISpot.3)To identify whether detected IL-17 secreting cells were Th17 cells or not, isolated CD4+T cells from 3 pSS patients who were positive for M3R specific IL-17 secreting cells, were co-cultured with auto-monocyte derived dendritic cells (DCs), and stimulated with the dominant IL-17 secreting M3R peptides detected in method 2.4)Anti-M3R antibodies were examined using ELISA method.5)Clinical features were compared between M3R specific Th17 cells positive and negative pSS patients.Results:1)5 of 10 (50%) pSS patients, while none of 10 (0%) HC, and 5 (0%) IgG4-RD patients, showed significantly increased IL-17 positive spots against selected M3R peptides mixed stimulation compared with non-stimulation in ELISpot (Figure 1). M3R specific IL-17 secreting cells were detected significantly more frequently in pSS (5/10, 50%) than in HC (0/10, 0%) (p=0.03).2)All 5 pSS patients, who were positive for M3R specific IL-17 secreting cells, showed significantly increased IL-17 positive spots against M3R AA76-95 peptides.3)Co-culturing CD4+ T cells with DCs, stimulated with identified dominant M3R peptides in method 2, showed significantly increased spots, clarifying that IL-17 secreting cells were peripheral M3R reactive Th17 cells.4)Titers of anti-M3R antibodies were significantly higher among M3R reactive Th17 cells positive pSS patients than negative pSS patients.5)5 pSS patients positive for M3R reactive Th17 cells had significantly higher disease activity score (ESSDAI: 8.0±4.3) than 5 negative pSS patients (2.8±1.7) (P=0.01).Conclusion:We detected circulating M3R reactive Th17 cells in pSS patients using ELISpot, whose T cell epitopes were shown to be included in M3R AA76-95. Moreover, M3R reactive Th17 cells might correlate with higher disease activity and production of anti-M3R antibodies in pSS patients.References:[1]Tsuboi H, et al. New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren’s syndrome.Clin Exp Immunol2010;162:53-61[2]Naito Y, et al. Altered peptide ligands regulate muscarinic acetylcholine receptor reactive T cells of patients with Sjögren’s syndrome.Ann Rheum Dis2005;65:269-71[3]Iizuka M, et al. Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren’s syndrome-like sialoadenitis.J Autoimmun.2010;35:383-9Disclosure of Interests:None declared
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Hering, Nina A., Verena Liu, Rayoung Kim, Benjamin Weixler, Raoul A. Droeser, Marco Arndt, Ioannis Pozios, Katharina Beyer, Martin E. Kreis, and Hendrik Seeliger. "Blockage of Cholinergic Signaling via Muscarinic Acetylcholine Receptor 3 Inhibits Tumor Growth in Human Colorectal Adenocarcinoma." Cancers 13, no. 13 (June 28, 2021): 3220. http://dx.doi.org/10.3390/cancers13133220.
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Cholinergic signaling via the muscarinic M3 acetylcholine receptor (M3R) is involved in the development and progression of colorectal cancer (CRC). The present study aimed to analyze the blocking of M3R signaling in CRC using darifenacin, a selective M3R antagonist. Darifenacin effects were studied on HT-29 and SW480 CRC cells using MTT and BrdU assays, Western blotting and real time RT-PCR. In vivo, blocking of M3R was assessed in an orthotopic CRC xenograft BALB/cnu/nu mouse model. M3R expression in clinical tumor specimens was studied by immunohistochemistry on a tissue microarray of 585 CRC patients. In vitro, darifenacin decreased tumor cell survival and proliferation in a dose-dependent manner. Acetylcholine-induced p38, ERK1/2 and Akt signaling, and MMP-1 mRNA expression were decreased by darifenacin, as well as matrigel invasion of tumor cells. In mice, darifenacin reduced primary tumor volume and weight (p < 0.05), as well as liver metastases, compared to controls. High expression scores of M3R were found on 89.2% of clinical CRC samples and correlated with infiltrative tumor border and non-mucinous histology (p < 0.05). In conclusion, darifenacin inhibited components of tumor growth and progression in vitro and reduced tumor growth in vivo. Its target, M3R, was expressed on the majority of CRC. Thus, repurposing darifenacin may be an attractive addition to systemic tumor therapy in CRC patients expressing M3R.
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Kawaguchi, Y., Y. Nakamura, I. Matsumoto, E. Nishimagi, T. Satoh, M. Kuwana, T. Sumida, and M. Hara. "Muscarinic-3 acetylcholine receptor autoantibody in patients with systemic sclerosis: contribution to severe gastrointestinal tract dysmotility." Annals of the Rheumatic Diseases 68, no. 5 (September 1, 2008): 710–14. http://dx.doi.org/10.1136/ard.2008.096545.
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Objective:Patients with systemic sclerosis (SSc) complicated by severe gastrointestinal tract (GIT) dysmotility at an early stage are difficult to treat and mortality is high. To clarify the pathogenesis of GIT involvement, the occurrence of autoantibody was investigated for muscarinic-3 acetylcholine receptor (M3R) in patients with SSc.Methods:Fourteen patients with severe GIT involvement (malabsorption syndrome and/or pseudo-obstruction) within 2 years of SSc onset (group 1) were enrolled in the present study. Sixty-two patients with SSc without severe GIT involvement within 2 years of onset (group 2) were also recruited, along with 70 healthy control subjects. Using an established enzyme immunoassay (EIA) system detecting autoantibody against the second loop domain of M3R, the presence of an anti-M3R antibody was examined in SSc patients.Results:The mean optical density (OD) titres of group 1 were significantly higher than those of group 2 (0.65 (SD 0.58) vs 0.066 (SD 0.13), p<0.001). The positivity of anti-M3R antibody was significantly higher in group 1 than in group 2 (9/14 vs 3/62, p = 2.5 × 10−6 by Fisher’s exact test). The cutoff OD was calculated from the EIA reaction of the 70 healthy controls (the mean value plus 2 SD was 0.295).Conclusion:The findings indicated that anti-M3R antibody very frequently appears in patients with SSc, which is accompanied by severe GIT involvement, suggesting that M3R-mediated enteric cholinergic neurotransmission may provide a pathogenic mechanism for GIT dysmotility in SSc.
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Rothstein, Joseph. "Korg M3R Synthesizer Module." Computer Music Journal 14, no. 4 (1990): 85. http://dx.doi.org/10.2307/3680802.
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Tolaymat, Mazen, Shannon Larabee, Shien Hu, Guofeng Xie, and Jean-Pierre Raufman. "The Role of M3 Muscarinic Receptor Ligand-Induced Kinase Signaling in Colon Cancer Progression." Cancers 11, no. 3 (March 5, 2019): 308. http://dx.doi.org/10.3390/cancers11030308.
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Despite a reduction in incidence over the past decade, colon cancer remains the second most common cause of cancer death in the United States; recent demographics suggest this disease is now afflicting younger persons. M3 muscarinic receptor (M3R) mRNA and protein are over-expressed in colon cancer, and M3R can be activated by both traditional (e.g., acetylcholine) and non-traditional (e.g., bile acids) muscarinic ligands. In this review, we weigh the data supporting a prominent role for key protein kinases downstream of M3R activation in promoting colon cancer progression and dissemination. Specifically, we explore the roles that downstream activation of the mitogen activated protein kinase/extracellular signal-related kinase (MAPK/ERK), protein kinase C, p38 MAPK, and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways play in mediating colon cancer cell proliferation, survival, migration and invasion. We assess the impact of M3R-stimulated induction of selected matrix metalloproteinases germane to these hallmarks of colon cancer progression. In this context, we also critically review the reproducibility of findings derived from a variety of in vivo and in vitro colon cancer models, and their fidelity to human disease. Finally, we summarize the therapeutic potential of targeting various steps from ligand-M3R interaction to the activation of key downstream molecules.
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Prayitno, Mohammad Agus, and Husna Amalana. "Pemberdayaan Siswa Madrasah Aliyah Melalui Pelatihan Chemoentrepreneurship Lilin Hias Aromaterapi." Dimas: Jurnal Pemikiran Agama untuk Pemberdayaan 19, no. 2 (November 28, 2019): 155. http://dx.doi.org/10.21580/dms.2019.192.5125.
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<p>Islamic Senior High School of Mu'allimin Mu'allimat Rembang (MA M3R) is one of the oldest school in Rembang Regency. Most of the students who attend the school are students from various Islamic boarding schools in the city of Rembang. Most of the graduates from MA M3R did not continue to Higher Education. There are those who work, get married, and those who continue chanting. For this reason, counseling activities are needed which are to provide training to MA M3R students who aim to equip entrepreneurial skills after graduation. One of the entrepreneurial skills that can be delivered is the making of aromatherapy decorative candles. The stages in the extension activities include 3 stages, namely preparation, implementation and evaluation. This service activity is able to increase student entrepreneurial motivation, train students' tenacity and responsibility, and train good ways of communicating when marketing product results.<strong></strong></p><p> </p><p>Madrasah Aliyah Mu’allimin Mu’allimat Rembang (MA M3R) merupakan salah satu madrasah tertua di Kabupaten Rembang. Sebagian besar siswa yang bersekolah di madarah tersebut adalah santri dari berbagai pondok pesantren yang ada di kota Rembang. Sebagian besar lulusan dari MA M3R tidak melanjutkan ke Perguruan Tinggi. Ada yang bekerja, menikah, dan adapula yang melanjutkan mengaji. Untuk itu diperlukan kegiatan penyuluhan yang sifatnya memberikan pelatihan terhadap siswa-siswi MA M3R yang bertujuan untuk membekali keterampilan wirausaha setelah lulus. Salah satu keterampilan wirausaha yang dapat disampaikan adalah pembuatan lilin hias beraromaterapi. Tahapan dalam kegiatan penyuluhan ini meliputi 3 tahap, yaitu persiapan, pelaksanaan, dan evaluasi. Kegiatan pengabdian ini mampu meningkatkan motivasi wirausaha siswa, melatih keuletan dan tanggung jawab siswa, serta melatih cara berkomunikasi yang baik pada saat memasarkan hasil produk.<strong></strong></p>
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Jarvis, Susan M., Ronald P. Morrissey, David J. Moretti, Nancy A. DiMarzio, and Jessica A. Shaffer. "Marine Mammal Monitoring on Navy Ranges (M3R): A Toolset for Automated Detection, Localization, and Monitoring of Marine Mammals in Open Ocean Environments." Marine Technology Society Journal 48, no. 1 (January 1, 2014): 5–20. http://dx.doi.org/10.4031/mtsj.48.1.1.
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AbstractNavy sonar has been associated with a number of marine mammal stranding events worldwide. As a result, determining the effects of anthropogenic noise on marine mammals is currently an active area of research. The development of methods to detect and localize the animals in their native environments is key to advancing this research and our understanding. This paper presents a collection of algorithms for automated passive acoustic detection, classification, and localization of vocalizing marine mammals in open ocean environments. The tool set known as M3R (Marine Mammal Monitoring on Navy Ranges) uses the large fields of wide-bandwidth bottom-mounted hydrophones that are part of the U.S. Navy’s undersea ranges to listen for vocalizing whales. M3R employs time-frequency analysis to passively detect whale vocalizations; it then aligns detections among neighboring hydrophones to determine the difference in times of arrival (TDOA) of each vocalization. Sets of TDOA are then used to determine 2-D or 3-D position points using hyperbolic localization techniques. An M3R system is capable of continuous, automated, real-time monitoring of over 200 wide-bandwidth hydrophones covering 2,000+ km2 of open ocean. M3R is typically used in a collaborative fashion to localize animals in support of tagging exercises, visual surveys, and behavioral response experiments.
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Mourad, Nizar I., Andrea Perota, Daela Xhema, Cesare Galli, and Pierre Gianello. "Transgenic Expression of Glucagon-Like Peptide-1 (GLP-1) and Activated Muscarinic Receptor (M3R) Significantly Improves Pig Islet Secretory Function." Cell Transplantation 26, no. 5 (May 2017): 901–11. http://dx.doi.org/10.3727/096368916x693798.
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Porcine islets show notoriously low insulin secretion levels in response to glucose stimulation. While this is somehow expected in the case of immature islets isolated from fetal and neonatal pigs, disappointingly low secretory responses are frequently reported in studies using in vitro-maturated fetal and neonatal islets and even fully differentiated adult islets. Herein we show that β-cell-specific expression of a modified glucagon-like peptide-1 (GLP-1) and of a constitutively activated type 3 muscarinic receptor (M3R) efficiently amplifies glucose-stimulated insulin secretion (GSIS). Both adult and neonatal isolated pig islets were treated with adenoviral expression vectors carrying sequences encoding for GLP-1 and/or M3R. GSIS from transduced and control islets was evaluated during static incubation and dynamic perifusion assays. While expression of GLP-1 did not affect basal or stimulated insulin secretion, activated M3R produced a twofold increase in both first and second phases of GSIS. Coexpression of GLP-1 and M3R caused an even greater increase in the secretory response, which was amplified fourfold compared to controls. In conclusion, our work highlights pig islet insulin secretion deficiencies and proposes concomitant activation of cAMP-dependent and cholinergic pathways as a solution to ameliorate GSIS from pig islets used for transplantation.
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Kuol, Nyanbol, Majid Davidson, Jimsheena Karakkat, Rhiannon T. Filippone, Margaret Veale, Rodney Luwor, Sarah Fraser, Vasso Apostolopoulos, and Kulmira Nurgali. "Blocking Muscarinic Receptor 3 Attenuates Tumor Growth and Decreases Immunosuppressive and Cholinergic Markers in an Orthotopic Mouse Model of Colorectal Cancer." International Journal of Molecular Sciences 24, no. 1 (December 29, 2022): 596. http://dx.doi.org/10.3390/ijms24010596.
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Tumor cells have evolved to express immunosuppressive molecules allowing their evasion from the host’s immune system. These molecules include programmed death ligands 1 and 2 (PD-L1 and PD-L2). Cancer cells can also produce acetylcholine (ACh), which plays a role in tumor development. Moreover, tumor innervation can stimulate vascularization leading to tumor growth and metastasis. The effects of atropine and muscarinic receptor 3 (M3R) blocker, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP), on cancer growth and spread were evaluated in vitro using murine colon cancer cell line, CT-26, and in vivo in an orthotopic mouse model of colorectal cancer. In the in vitro model, atropine and 4-DAMP significantly inhibited CT-26 cell proliferation in a dose dependent manner and induced apoptosis. Atropine attenuated immunosuppressive markers and M3R via inhibition of EGFR/AKT/ERK signaling pathways. However, 4-DAMP showed no effect on the expression of PD-L1, PD-L2, and choline acetyltransferase (ChAT) on CT-26 cells but attenuated M3R by suppressing the phosphorylation of AKT and ERK. Blocking of M3R in vivo decreased tumor growth and expression of immunosuppressive, cholinergic, and angiogenic markers through inhibition of AKT and ERK, leading to an improved immune response against cancer. The expression of immunosuppressive and cholinergic markers may hold potential in determining prognosis and treatment regimens for colorectal cancer patients. This study’s results demonstrate that blocking M3R has pronounced antitumor effects via several mechanisms, including inhibition of immunosuppressive molecules, enhancement of antitumor immune response, and suppression of tumor angiogenesis via suppression of the AKT/ERK signaling pathway. These findings suggest a crosstalk between the cholinergic and immune systems during cancer development. In addition, the cholinergic system influences cancer evasion from the host’s immunity.
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Musayeva, Aytan, Subao Jiang, Yue Ruan, Jenia Kouchek Zadeh, Panagiotis Chronopoulos, Norbert Pfeiffer, Werner E. G. Müller, et al. "Aged Mice Devoid of the M3 Muscarinic Acetylcholine Receptor Develop Mild Dry Eye Disease." International Journal of Molecular Sciences 22, no. 11 (June 7, 2021): 6133. http://dx.doi.org/10.3390/ijms22116133.
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The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1–M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R−/−). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R−/− mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R−/− mice. M3R−/− mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M3 receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.
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Cheng, Kunrong, Roxana Samimi, Guofeng Xie, Jasleen Shant, Cinthia Drachenberg, Mark Wade, Richard J. Davis, George Nomikos, and Jean-Pierre Raufman. "Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation." American Journal of Physiology-Gastrointestinal and Liver Physiology 295, no. 3 (September 2008): G591—G597. http://dx.doi.org/10.1152/ajpgi.00055.2008.
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Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist ( p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by ∼40% ( P < 0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively ( P < 0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes ( n = 25) whereas half of colon cancer specimens ( n = 24) exhibited moderate to strong staining ( P < 0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.
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McLean, Leon P., Allen Smith, Lumei Cheung, Joseph F. Urban, Rex Sun, Viktoriya Grinchuk, Neemesh Desai, Aiping Zhao, Jean-Pierre Raufman, and Terez Shea-Donohue. "Type 3 muscarinic receptors contribute to intestinal mucosal homeostasis and clearance of Nippostrongylus brasiliensis through induction of TH2 cytokines." American Journal of Physiology-Gastrointestinal and Liver Physiology 311, no. 1 (July 1, 2016): G130—G141. http://dx.doi.org/10.1152/ajpgi.00461.2014.
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Despite increased appreciation for the role of nicotinic receptors in the modulation of and response to inflammation, the contribution of muscarinic receptors to mucosal homeostasis, clearance of enteric pathogens, and modulation of immune cell function remains relatively undefined. Uninfected and Nippostrongylus brasiliensis-infected wild-type and type 3 muscarinic receptor (M3R)-deficient ( Chrm3 −/−) mice were studied to determine the contribution of M3R to mucosal homeostasis as well as host defense against the TH2-eliciting enteric nematode N. brasiliensis. Intestinal permeability and expression of TH1/TH17 cytokines were increased in uninfected Chrm3 −/− small intestine. Notably, in Chrm3 −/− mice infected with N. brasiliensis, small intestinal upregulation of TH2 cytokines was attenuated and nematode clearance was delayed. In Chrm3 −/− mice, TH2-dependent changes in small intestinal function including smooth muscle hypercontractility, increased epithelial permeability, decreased epithelial secretion and absorption, and goblet cell expansion were absent despite N. brasiliensis infection. These findings identify an important role for M3R in host defense and clearance of N. brasiliensis, and support the expanding role of cholinergic muscarinic receptors in maintaining mucosal homeostasis.
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Yang, Baofeng, Yanjie Lu, and Jundong Jiao. "A novel antiarrhythmic target: M3R/IKM3." Journal of Molecular and Cellular Cardiology 42, no. 6 (June 2007): S13. http://dx.doi.org/10.1016/j.yjmcc.2007.03.036.
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Schegg, Vanessa, Monique Vogel, Svetlana Didichenko, Michael B. Stadler, Zsuzsanna Beleznay, Stephan Gadola, Christine Sengupta, Beda M. Stadler, and Sylvia M. Miescher. "Evidence that anti-muscarinic antibodies in Sjögren's syndrome recognise both M3R and M1R." Biologicals 36, no. 4 (July 2008): 213–22. http://dx.doi.org/10.1016/j.biologicals.2007.11.001.
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Harrington, Andrea M., Cristal J. Peck, Lu Liu, Elizabeth Burcher, John M. Hutson, and Bridget R. Southwell. "T1758 Immunohistochemical Localisation Shows That Muscarinic Receptors M1R, M2R and M3R Mediate Both Cholinergic and Nitrergic Pathways in Human Colon." Gastroenterology 138, no. 5 (May 2010): S—572. http://dx.doi.org/10.1016/s0016-5085(10)62636-4.
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Kistemaker, Loes E. M., Ronald P. van Os, Albertina Dethmers-Ausema, I. Sophie T. Bos, Machteld N. Hylkema, Maarten van den Berge, Pieter S. Hiemstra, et al. "Muscarinic M3 receptors on structural cells regulate cigarette smoke-induced neutrophilic airway inflammation in mice." American Journal of Physiology-Lung Cellular and Molecular Physiology 308, no. 1 (January 1, 2015): L96—L103. http://dx.doi.org/10.1152/ajplung.00259.2014.
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Anticholinergics, blocking the muscarinic M3 receptor, are effective bronchodilators for patients with chronic obstructive pulmonary disease. Recent evidence from M3 receptor-deficient mice (M3R−/−) indicates that M3 receptors also regulate neutrophilic inflammation in response to cigarette smoke (CS). M3 receptors are present on almost all cell types, and in this study we investigated the relative contribution of M3 receptors on structural cells vs. inflammatory cells to CS-induced inflammation using bone marrow chimeric mice. Bone marrow chimeras (C56Bl/6 mice) were generated, and engraftment was confirmed after 10 wk. Thereafter, irradiated and nonirradiated control animals were exposed to CS or fresh air for four consecutive days. CS induced a significant increase in neutrophil numbers in nonirradiated and irradiated control animals (4- to 35-fold). Interestingly, wild-type animals receiving M3R−/− bone marrow showed a similar increase in neutrophil number (15-fold). In contrast, no increase in the number of neutrophils was observed in M3R−/− animals receiving wild-type bone marrow. The increase in keratinocyte-derived chemokine (KC) levels was similar in all smoke-exposed groups (2.5- to 5.0-fold). Microarray analysis revealed that fibrinogen-α and CD177, both involved in neutrophil migration, were downregulated in CS-exposed M3R−/− animals receiving wild-type bone marrow compared with CS-exposed wild-type animals, which was confirmed by RT-qPCR (1.6–2.5 fold). These findings indicate that the M3 receptor on structural cells plays a proinflammatory role in CS-induced neutrophilic inflammation, whereas the M3 receptor on inflammatory cells does not. This effect is probably not mediated via KC release, but may involve altered adhesion and transmigration of neutrophils via fibrinogen-α and CD177.
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D Cu-Quiñones, Luis, Manuel J. Chan-Bacab, Carlos Granados-Echagoyen, Benjamin O. Ortega Morales, Pedro Zamora-Crescencio, Emanuel Hernández-Núñez, Diana Lizbeth Gomez-Galicia, and Francisco J. Aguirre-Crespo. "Taraxasterol presente en hojas de Jatropha gaumeri Greenm, como teórico antagonista parcial del receptor muscarínico Acho-M;, del tracto gastrointestinal." Quimica Hoy 10, no. 3 (January 10, 2022): 7–11. http://dx.doi.org/10.29105/qh10.3-263.
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Jatropha gaumeri es un árbol endémico de la Península de Yucatán, México, y cuenta con estudios etnomédicos,fitoquimicos y farmacológicos. En el presente trabajo se plantea generar información teórica que relacione las propiedadesfisicoquímicas y biológicas con los efectos atribuidos en la medicina tradicional y certidumbre en el desarrollo de estudiosexperimentales. J. gaumeri se analizó por gravimetria y espectroscopia (UV-Vis, FTIR) y taraxasterol (6) medianteMolinspiration, Osiris y AutoDock Vina para el acoplamiento con el receptor muscarínico tipo 3 (Acho-M3r). Se registraun rendimiento del 14.66 ± 0.75%, por UV-Vis se identifican bandas relacionadas a compuestos fenólicos y flavonoides;por FTIR se identifican bandas relacionadas con 6. Por otro lado, 6 presenta una violación a la Regla de Lipinski,interacción con receptores nucleares, enzimas y con receptores acoplados a proteinas G, no se reportan riesgos demutagenicidad, tumorogenicidad, irritabilidad y sobre reproducción. 6 presenta alta probabilidad de absorción en tractogastrointestinal (TGD, inhibición de OATP1B1, BSEP y hERG, sustrato del CYP3A4 así como de receptores aestrógenos. El sitio activo del Acho-M3r se optimizó con escopolamina (RMSD: 2.48 Á) y 6 registro una alta afinidad conel Acho-M3r, pero menor a la de escopolamina (-10.18 vs.-5.9 Kcal/mol); los valores de Ki (0.03 vs. 0.48 uM) presentanel mismo comportamiento, hechos que sugieren interacción parcial mediante fuerzas de Van der Waals. Bajo estecontexto, 6 presenta buena absorción, baja permeabilidad, potencial acumulación en la superficie apical del enterocito,interacción con los Acho-M3r favoreciendo la inducción de efectos a nivel del TGI, modificación de la biodisponibilidady la farmacocinética de fármacos empleados de manera simultánea con las preparaciones tradicionales de J. gaumeri,entre otras especies vegetales medicinales. Se requieres evaluaciones experimentales (in vitro, ex vivo, in vivo) quecorroboren el planteamiento.
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Mona, Mahmoud, Rehae Miller, Hui Li, Yun-Jong Park, Raafi Zaman, Li-Jun Yang, and Seunghee Cha. "MIST1, an Inductive Signal for Salivary Amylase in Mesenchymal Stem Cells." International Journal of Molecular Sciences 20, no. 3 (February 12, 2019): 767. http://dx.doi.org/10.3390/ijms20030767.
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Sjögren’s syndrome (SjS) is an autoimmune disease that destroys the salivary glands and results in severe dry mouth. Mesenchymal stem cell (MSC) transplantation has been recently proposed as a promising therapy for restoring cells in multiple degenerative diseases. We have recently utilized advanced proteomics biochemical assays to identify the key molecules involved in the mesenchymal-epithelial transition (MET) of co-cultured mouse bone-marrow-derived MSCs mMSCs with primary salivary gland cells. Among the multiple transcription factors (TFs) that were differentially expressed, two major TFs were selected: muscle, intestine, and stomach expression-1 (MIST1) and transcription factor E2a (TCF3). These factors were assessed in the current study for their ability to drive the expression of acinar cell marker, alpha-salivary amylase 1 (AMY1), and ductal cell marker, cytokeratin19 (CK19), in vitro. Overexpression of MIST1-induced AMY1 expression while it had little effect on CK19 expression. In contrast, TCF3 induced neither of those cellular markers. Furthermore, we have identified that mMSCs express muscarinic-type 3 receptor (M3R) mainly in the cytoplasm and aquaporin 5 (AQP5) in the nucleus. While MIST1 did not alter M3R levels in mMSCs, a TCF3 overexpression downregulated M3R expressions in mMSCs. The mechanisms for such differential regulation of glandular markers by these TFs warrant further investigation.
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Adrian, Odile. "M3R AESA technology for extended air defence." IEEE Aerospace and Electronic Systems Magazine 25, no. 8 (August 2010): 11–16. http://dx.doi.org/10.1109/maes.2010.5552607.
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Larabee, Shannon M., Kunrong Cheng, Jean-Pierre Raufman, and Shien Hu. "Muscarinic receptor activation in colon cancer selectively augments pro-proliferative microRNA-21, microRNA-221 and microRNA-222 expression." PLOS ONE 17, no. 6 (June 3, 2022): e0269618. http://dx.doi.org/10.1371/journal.pone.0269618.
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Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.
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Ali, Osman, Mazen Tolaymat, Shien Hu, Guofeng Xie, and Jean-Pierre Raufman. "Overcoming Obstacles to Targeting Muscarinic Receptor Signaling in Colorectal Cancer." International Journal of Molecular Sciences 22, no. 2 (January 13, 2021): 716. http://dx.doi.org/10.3390/ijms22020716.
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Despite great advances in our understanding of the pathobiology of colorectal cancer and the genetic and environmental factors that mitigate its onset and progression, a paucity of effective treatments persists. The five-year survival for advanced, stage IV disease remains substantially less than 20%. This review examines a relatively untapped reservoir of potential therapies to target muscarinic receptor expression, activation, and signaling in colorectal cancer. Most colorectal cancers overexpress M3 muscarinic receptors (M3R), and both in vitro and in vivo studies have shown that activating these receptors stimulates cellular programs that result in colon cancer growth, survival, and spread. In vivo studies using mouse models of intestinal neoplasia have shown that using either genetic or pharmacological approaches to block M3R expression and activation, respectively, attenuates the development and progression of colon cancer. Moreover, both in vitro and in vivo studies have shown that blocking the activity of matrix metalloproteinases (MMPs) that are induced selectively by M3R activation, i.e., MMP1 and MMP7, also impedes colon cancer growth and progression. Nonetheless, the widespread expression of muscarinic receptors and MMPs and their importance for many cellular functions raises important concerns about off-target effects and the safety of employing similar strategies in humans. As we highlight in this review, highly selective approaches can overcome these obstacles and permit clinicians to exploit the reliance of colon cancer cells on muscarinic receptors and their downstream signal transduction pathways for therapeutic purposes.
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Sumida, Takayuki, Hiroto Tsuboi, Mana Iizuka, Yumi Nakamura, and Isao Matsumoto. "Functional role of M3 muscarinic acetylcholine receptor (M3R) reactive T cells and anti-M3R autoantibodies in patients with Sjögren's syndrome." Autoimmunity Reviews 9, no. 9 (July 2010): 615–17. http://dx.doi.org/10.1016/j.autrev.2010.05.008.
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Yang, Lin, Jinzhe Ju, Wei Zhang, Fengfeng Lv, Chunyan Pang, Guoan Yang, and Yongfu Wang. "Effects of Muscarinic Acetylcholine 3 Receptor208-227Peptide Immunization on Autoimmune Response in Nonobese Diabetic Mice." Clinical and Developmental Immunology 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/485213.
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The second extracellular loop (LFWQYFVGKRTVPPGECFIQFLSEPTITFGTAI, aa 205–237) of muscarinic acetylcholine 3 receptor (M3R) has been reported to be an epitope for autoantibodies generated during certain autoimmune disorders, including Sjögren’s syndrome (SS). Autoantibodies against M3R228–237have been shown to interfere with the function of M3R. However, few studies have been performed on the M3R205–227peptide of the second extracellular loop. In the current study, we sought to investigate the effect of M3R208–227peptide immunization on autoimmune response in NOD/LtJ mice. We synthesized the M3R208–227peptide and immunized NOD/LtJ mice to investigate whether peptide-specific antibodies could be generated and whether immunization would lead to changes in autoimmune response in NOD/LtJ mice. Our results demonstrate that the secretions of Th-1, Th-2, and Th-17 cytokines are downregulated and lymphocytic infiltration is improved in the salivary glands and lacrimal glands following immunization with M3R208–227peptide in NOD/LtJ mice, suggesting that peptide immunotherapy using the M3R208–227peptide may represent a potential therapeutic alternative.
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Guareschi, Zoé M., Ana C. Valcanaia, Vanessa M. Ceglarek, Pamela Hotz, Bruna K. Amaral, Domwesley W. de Souza, Thainan A. de Souza, et al. "The effect of chronic oral vitamin D supplementation on adiposity and insulin secretion in hypothalamic obese rats." British Journal of Nutrition 121, no. 12 (June 13, 2019): 1334–44. http://dx.doi.org/10.1017/s0007114519000667.
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AbstractReduced plasma vitamin D (VD) levels may contribute to excessive white adipose tissue, insulin resistance (IR) and dyslipidaemia. We evaluated the effect of chronic oral VD supplementation on adiposity and insulin secretion in monosodium glutamate (MSG)-treated rats. During their first 5 d of life, male neonate rats received subcutaneous injections of MSG (4 g/kg), while the control (CON) group received saline solution. After weaning, groups were randomly distributed into VD supplemented (12 µg/kg; three times/week) and non-supplemented (NS) rats, forming four experimental groups (n 15 rats/group): CON-NS, CON-VD, MSG-NS and MSG-VD. At 76 d of life, rats were submitted to an oral glucose tolerance test (OGTT; 2 g/kg), and at 86 d, obesity, IR and plasma metabolic parameters were evaluated. Pancreatic islets were isolated for glucose-induced insulin secretion (GIIS), cholinergic insulinotropic response and muscarinic 3 receptor (M3R), protein kinase C (PKC) and protein kinase A (PKA) expressions. Pancreas was submitted to histological analyses. VD supplementation decreased hyperinsulinaemia (86 %), hypertriacylglycerolaemia (50 %) and restored insulin sensibility (89 %) in MSG-VD rats, without modifying adiposity, OGTT or GIIS, compared with the MSG-NS group. The cholinergic action was reduced (57 %) in islets from MSG-VD rats, without any change in M3R, PKA or PKC expression. In conclusion, chronic oral VD supplementation of MSG-obese rats was able to prevent hyperinsulinaemia and IR, improving triacylglycerolaemia without modifying adiposity. A reduced cholinergic pancreatic effect, in response to VD, could be involved in the normalisation of plasma insulin levels, an event that appears to be independent of M3R and its downstream pathways.
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Said, Anan H., Shien Hu, Ameer Abutaleb, Tonya Watkins, Kunrong Cheng, Ahmed Chahdi, Panjamurthy Kuppusamy, Neeraj Saxena, Guofeng Xie, and Jean-Pierre Raufman. "Interacting post-muscarinic receptor signaling pathways potentiate matrix metalloproteinase-1 expression and invasion of human colon cancer cells." Biochemical Journal 474, no. 5 (February 20, 2017): 647–65. http://dx.doi.org/10.1042/bcj20160704.
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M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 (MMP1) expression. MMP1 expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced MMP1 expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/β alone attenuated, but did not abolish ACh-induced MMP1 expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced MMP1 expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/β inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated MMP1 gene and protein expression, and cell invasion. PMA- and ACh-induced MMP1 expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/β and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment MMP1 expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.
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Patowary, Suparna, Elisa Alvarez-Curto, Tian-Rui Xu, Jessica D. Holz, Julie A. Oliver, Graeme Milligan, and Valerică Raicu. "The muscarinic M3 acetylcholine receptor exists as two differently sized complexes at the plasma membrane." Biochemical Journal 452, no. 2 (May 10, 2013): 303–12. http://dx.doi.org/10.1042/bj20121902.
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The literature on GPCR (G-protein-coupled receptor) homo-oligomerization encompasses conflicting views that range from interpretations that GPCRs must be monomeric, through comparatively newer proposals that they exist as dimers or higher-order oligomers, to suggestions that such quaternary structures are rather ephemeral or merely accidental and may serve no functional purpose. In the present study we use a novel method of FRET (Förster resonance energy transfer) spectrometry and controlled expression of energy donor-tagged species to show that M3Rs (muscarinic M3 acetylcholine receptors) at the plasma membrane exist as stable dimeric complexes, a large fraction of which interact dynamically to form tetramers without the presence of trimers, pentamers, hexamers etc. That M3R dimeric units interact dynamically was also supported by co-immunoprecipitation of receptors synthesized at distinct times. On the basis of all these findings, we propose a conceptual framework that may reconcile the conflicting views on the quaternary structure of GPCRs.
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Delvalle, Ninotchska M., David E. Fried, Gretchen Rivera-Lopez, Luke Gaudette, and Brian D. Gulbransen. "Cholinergic activation of enteric glia is a physiological mechanism that contributes to the regulation of gastrointestinal motility." American Journal of Physiology-Gastrointestinal and Liver Physiology 315, no. 4 (October 1, 2018): G473—G483. http://dx.doi.org/10.1152/ajpgi.00155.2018.
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The reflexive activities of the gastrointestinal tract are regulated, in part, by precise interactions between neurons and glia in the enteric nervous system (ENS). Intraganglionic enteric glia are a unique type of peripheral glia that surround enteric neurons and regulate neuronal function, activity, and survival. Enteric glia express numerous neurotransmitter receptors that allow them to sense neuronal activity, but it is not clear if enteric glia monitor acetylcholine (ACh), the primary excitatory neurotransmitter in the ENS. Here, we tested the hypothesis that enteric glia detect ACh and that glial activation by ACh contributes to the physiological regulation of gut functions. Our results show that myenteric enteric glia express both the M3 and M5 subtypes of muscarinic receptors (MRs) and that muscarine drives intracellular calcium (Ca2+) signaling predominantly through M3R activation. To elucidate the functional effects of activation of glial M3Rs, we used GFAP::hM3Dq mice that express a modified human M3R (hM3Dq) exclusively on glial fibrillary acidic protein (GFAP) positive glia to directly activate glial hM3Dqs using clozapine- N-oxide. Using spatiotemporal mapping analysis, we found that the activation of glial hM3Dq receptors enhances motility reflexes ex vivo. Continuous stimulation of hM3Dq receptors in vivo, drove changes in gastrointestinal motility without affecting neuronal survival in the ENS and glial muscarinic receptor activation did not alter neuron survival in vitro. Our results provide the first evidence that GFAP intraganglionic enteric glia express functional muscarinic receptors and suggest that the activation of glial muscarinic receptors contributes to the physiological regulation of functions. NEW & NOTEWORTHY Enteric glia are emerging as novel regulators of enteric reflex circuits, but little is still known regarding the effects of specific transmitter pathways on glia and the resulting consequences on enteric reflexes. Here, we provide the first evidence that enteric glia monitor acetylcholine in the enteric nervous system and that glial activation by acetylcholine is a physiological mechanism that contributes to the functional regulation of intestinal reflexes.
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-LENOIR, Michel. "Le M3R, un concept de radar mobile, multifonction, et modulaire." Revue de l'Electricité et de l'Electronique -, no. 01 (2000): 73. http://dx.doi.org/10.3845/ree.2000.007.
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Khurana, Sandeep, Nirish Shah, William S. Twaddell, Jennifer A. Timmons, Xue-Min Gao, Kunrong Cheng, and Jean-Pierre Raufman. "S1880 Divergent Effects of M1 (M1r) and M3 (M3r) Muscarinic Receptor Deletion in Azoxymethane (AOM)-Induced Liver Injury." Gastroenterology 138, no. 5 (May 2010): S—808—S—809. http://dx.doi.org/10.1016/s0016-5085(10)63726-2.
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Fayon, Michael, Eric Dumas De La Roque, Patrick Berger, Hugues Begueret, Olga Ousova, Mathieu Molimard, and Roger Marthan. "Increased relaxation of immature airways to β2-adrenoceptor agonists is related to attenuated expression of postjunctional smooth muscle muscarinic M2 receptors." Journal of Applied Physiology 98, no. 4 (April 2005): 1526–33. http://dx.doi.org/10.1152/japplphysiol.00948.2004.
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Spontaneous or agonist-induced contraction of airway smooth muscle can be observed very early in fetal life, thus explaining the possible occurrence of bronchospasm in very low birth weight infants within the first days of life. In an attempt to better manage such bronchospasms, the aim of the present study was to investigate the age-specific modifications in airway smooth muscle relaxation to β2-agonists and muscarinic antagonists using a combination of functional and molecular techniques. In the rat, isometric relaxation to the β2-agonist salbutamol was examined in tracheae; we also examined muscarinic receptor expression (M2R and M3R mRNA levels) in airway smooth muscle by immunochemistry, Western blotting, and real-time PCR. Compared with adults, salbutamol-induced relaxation was twofold greater in immature rat isolated tracheae preconstricted by carbachol. This effect was associated with a lower expression of M2R in the smooth muscle of immature animals (sixfold and almost twofold as assessed by immunochemistry and Western blotting, respectively). Real-time PCR data indicate that changes in M2R expression according to age occurred at a posttranscriptional level. In adult airways, there was a significantly greater functional efficacy of M2R blockade by methoctramine compared with that shown in immature rats. Because of the limited availability of human neonate lung tissue, only the molecular part of the study was performed, and we observed a qualitatively similar effect, i.e., a lower M2R expression in the neonatal airway smooth muscle, although this was quantitatively smaller. We conclude that β2-agonist-induced relaxation is enhanced in immature compared with adult airways as a result of greater postjunctional M2R expression in adult airway smooth muscle. This finding may be of importance in the clinical management of bronchoconstriction in neonates.
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Zhao, Cuimei, Jie Qi, Xingyuan Liu, Huaizhi Chen, Jun Li, Yi Liu, Lei Liang, et al. "Refractoriness of the Sheep Superior Vena Cava Myocardial Sleeve." Experimental Biology and Medicine 233, no. 11 (November 2008): 1441–47. http://dx.doi.org/10.3181/0803-rm-102.
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The cardiomyocytes in the superior vena cava (SVC) myocardial sleeve have distinct action potentials and ionic current profiles, but the refractoriness of these cells has not been reported. Using standard intracellular microelectrode techniques, we demonstrated in sheep that the effective refractory period (ERP) of the cardiomyocytes in the SVC (114.7 ± 6.5 ms) is shorter than that in the inferior vena cava (IVC) (166.7 ± 6.2 ms), right atrial free wall (RAFW) (201.0 ± 6.0 ms) and right atrial appendage (RAA) (203.1 ± 5.8 ms) ( P < 0.05). The right atrial cardiomyocyte ERP was heterogeneously shortened by acetylcholine, a muscarinic type 2 receptor (M2R) agonist. After perfusion with 15 μM acetylcholine, the shortest ERP occurred in the SVC (the ERP in the SVC, IVC, RAFW and RAA was 53.6 ± 2.7, 98.9 ± 2.2, 121.8 ± 6.0 and 109.7 ± 5.1 ms, respectively; P < 0.05). Carbachol (1 μM), another M2R agonist, produced a similar effect as acetylcholine. Furthermore, we used methoctramine, a M2R blocker, 4-DAMP, a muscarinic type 3 receptor (M3R) blocker, and tropicamide, a muscarinic type 4 receptor (M4R) blocker to inhibit the acetylcholine-induced ERP shortening of SVC cardiomyocytes, and found that the 50% inhibitory concentration for methoctramine, 4-DAMP and tropicamide was 5.91, 45.72 and 80.34 nM, respectively. Therefore, we conclude that the sheep SVC myocardial sleeve is a unique electrophysiological region of the right atrium with the shortest ERP both under physiological condition and under cholinergic agonist stimulation. M2R might play a major role in the response of the SVC myocardial sleeve to parasympathetic nerve tone. The association between the distinct refractoriness in SVC and atrial fibrillation originating from the region deserves further investigation.
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Audi, S. H., G. S. Krenz, J. H. Linehan, D. A. Rickaby, and C. A. Dawson. "Pulmonary capillary transport function from flow-limited indicators." Journal of Applied Physiology 77, no. 1 (July 1, 1994): 332–51. http://dx.doi.org/10.1152/jappl.1994.77.1.332.
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The objective of this study was to examine the use of rapidly diffusing (flow-limited) indicators for estimating the pulmonary capillary blood volume (i.e., fraction of the lung blood volume wherein the diffusible indicators equilibrate with the tissue) and the capillary transit time distribution. Supporting theory and an application to experimental data are presented. The theory leads to the following equations, which relate the mean transit time (t), the variance (sigma 2), and the third central moment (m3) of the capillary transport function, hc(t), to the moments of the venous concentration-time curves for a vascular reference indicator, CR(t), and a flow-limited diffusible indicator, CD(t), after a bolus injection of the indicators upstream from an organ: sigma 2D - sigma 2R = ([1 + (te/tc)]2–1)sigma 2c and m3D-m3R = ([1 + (te/tc)]3–1)m3c, where te = tD - tR and tc is capillary t. The moments of hc(t) can be estimated if the injected bolus includes, along with the vascular reference indicator, at least two flow-limited diffusible indicators, each with a different te. A least-squares optimization procedure can then be used to specify the moments of hc(t). This approach was applied to isolated dog lung lobes with [14C]-diazepam as the diffusible indicator. The tissue-to-perfusate partition coefficient for [14C]diazepam could be adjusted to any desired value by altering the perfusate albumin concentration. Thus, by making a number of injections, each at a different perfusate albumin concentration, data were obtained in a manner equivalent to making one injection with a number of flow-limited diffusible indicators, each with a different te. On average, the estimated capillary volume and mean transit time were approximately 48% of the total lobar volume and mean transit time, and the relative dispersion of the hc(t) was approximately 75%.