M1 Acetylcholine Receptors Research Articles

Synergistic regulation mechanism of iperoxo and LY2119620 for muscarinic acetylcholine M2 receptor.

Muscarinic acetylcholine receptors are GPCRs that regulate the activity of a diverse array of central and peripheral functions in the human body, including the parasympathetic actions of acetylcholine. The M2 muscarinic receptor subtype plays a key role in modulating cardiac function and many important central processes. The orthosteric agonist and allosteric modulator can bind the pocket of M2. However, the detailed relationship between orthosteric agonist and allosteric modulator of M2 is still unclear. In this study, we intend to elucidate the residue-level regulation mechanism and pathway via a combined approach of dynamical correlation network and molecular dynamics simulation. Specifically computational residue-level fluctuation correlation data was analyzed to reveal detailed dynamics signatures in the regulation process. A hypothesis of “synergistic regulation” is proposed to reveal the cooperation affection between the orthosteric agonist and allosteric modulator, which is subsequently validated by perturbation and mutation analyses. Two possible synergistic regulation pathways of 2CU-I178-Y403-W400-F396-L114-Y440-Nb9 and IXO-V111-F396-L114-Y440-Nb9 were identified by the shortest path algorithm and were confirmed by the mutation of junction node. Furthermore, the efficiency of information transfer of bound M2 is significant higher than any single binding system. Our study shows that targeting the synergistic regulation pathways may better regulate the calcium channel of M2. The knowledge gained in this study may help develop drugs for diseases of the central nervous system and metabolic disorders.

Quercetin promotes gastrointestinal motility and mucin secretion in loperamide-induced constipation of SD rats through regulation of the mAChRs downstream signal

Context: Quercetin (QCT) has been known as a potential therapeutic strategy for gastrointestinal diseases because it contributes to the stabilization of mast cells, the prevention of histamine release and modulation of CaCC chloride channel.Objective: We investigated the laxative effect and action mechanism of QCT in Lop-induced constipation model.Materials and methods: Constipation of SD rats was induced by subcutaneous injection of loperamide (Lop) (4 mg/kg weight) in 0.5% Tween 20 twice a day for three days. After 24 h, the constipation group was further treated with 1× PBS (Lop + Vehicle treated group), 10 mg/kg of QCT (Lop + LQCT treated group), 20 mg/kg of QCT (Lop + MQCT treated group) or 40 mg/kg QCT (Lop + HQCT treated group) at once. At 24 h after QCT treatment, the constipation phenotypes were measured and the transverse colon was collected from SD rats.Results: The gastrointestinal motility, the number of stools and histological structures were significantly recovered in Lop + QCT treated group compared with the Lop + Vehicle treated group. Also, above activity of epithelial cells and smooth muscle cells were regulated by the mRNA expression of the muscarinic acetylcholine receptors M2 and M3 (mAChR M2 and M3) and some mediators of their downstream signalling pathway. Finally, laxative effects of QCT on mAChR signalling pathway were significantly inhibited by the treatment of mAChR antagonist in primary smooth muscle of rat intestine cells (pRISMCs).Conclusions: This study provides the first strong evidence that QCT can be considered an important candidate for improving chronic constipation induced by Lop treatment in animal models.

Presynaptic modulation of GABAergic transmission onto striatal cholinergic interneuron by M1 acetylcholine receptor activation

Presynaptic modulation of GABAergic transmission onto striatal cholinergic interneuron by M1 acetylcholine receptor activation

Muscarinic acetylcholine receptor M1 mediates prostate cancer cell migration and invasion through hedgehog signaling.

The autonomic nervous system contributes to prostate cancer proliferation and metastasis. However, the exact molecular mechanism remains unclear. In this study, muscarinic acetylcholine receptor M1 (CHRM1) expression was measured via immunohistochemical analysis in human prostate cancer tissue array slides. PC-3, LNCaP, and A549 cells were treated with pirenzepine or carbachol, and the cell migration and invasion abilities were evaluated. Western blotting and quantitative real-time PCR were performed to measure GLI family zinc finger 1 (GLI1), patched 1 (PTCH1), and sonic hedgehog (SHH) expression levels. High expression of CHRM1 was found in early-stage human prostate cancer tissues. In addition, the selective CHRM1 antagonist pirenzepine inhibited PC-3, LNCaP, and A549 cell migration and invasion, but the agonist carbachol promoted the migration and invasion of these three cell lines. Muscarinic signaling can be relayed by hedgehog signaling. These data show that CHRM1 is involved in the regulation of prostate cancer migration and invasion through the hedgehog signaling pathway.

Effects of muscarinic M1 and M4 acetylcholine receptor stimulation on extinction and reinstatement of cocaine seeking in male mice, independent of extinction learning.

Stimulating muscarinic M1/M4 receptors can blunt reinforcing and other effects of cocaine. A hallmark of addiction is continued drug seeking/craving after abstinence and relapse. We tested whether stimulating M1 and/or M4 receptors could facilitate extinction of cocaine seeking, and whether this was mediated via memory consolidation. Experimentally naïve C57BL/6J mice were allowed to acquire self-administration of intravenous cocaine (1mg/kg/infusion) under a fixed-ratio 1 schedule of reinforcement. Then, saline was substituted for cocaine until responding extinguished to ≤30% of cocaine-reinforced responding. Immediately after each extinction session, mice received saline, the M1/M4 receptor-preferring agonist xanomeline, the M1 receptor-selective allosteric agonist VU0357017, the M4 receptor-selective positive allosteric modulator VU0152100, or VU0357017+VU0152100. In additional experiments, xanomeline was administered delayed after the session or in the home cage before extinction training began. In the latter group, reinstatement of responding by a 10-mg/kg cocaine injection was also tested. Stimulating M1+M4 receptors significantly expedited extinction from 17.2 sessions to 8.3 using xanomeline or 7.8 using VU0357017+VU0152100. VU0357017 alone and VU0152100 alone did not significantly modify rates of extinction (12.6 and 14.6 sessions). The effect of xanomeline was fully preserved when administered delayed after or unpaired from extinction sessions (7.5 and 6.4 sessions). Xanomeline-treated mice showed no cocaine-induced reinstatement. These findings show that M1/M4 receptor stimulation can decrease cocaine seeking in mice. The effect lasted beyond treatment duration and was not dependent upon extinction learning. This suggests that M1/M4 receptor stimulation modulated or reversed some neurochemical effects of cocaine exposure.

Oxotremorine-M potentiates NMDA receptors by muscarinic receptor dependent and independent mechanisms

Muscarinic acetylcholine M1 receptors play an important role in synaptic plasticity in the hippocampus and cortex. Potentiation of NMDA receptors as a consequence of muscarinic acetylcholine M1 receptor activation is a crucial event mediating the cholinergic modulation of synaptic plasticity, which is a cellular mechanism for learning and memory. In Alzheimer's disease, the cholinergic input to the hippocampus and cortex is severely degenerated, and agonists or positive allosteric modulators of M1 receptors are therefore thought to be of potential use to treat the deficits in cognitive functions in Alzheimer's disease. In this study we developed a simple system in which muscarinic modulation of NMDA receptors can be studied in vitro. Human M1 receptors and NR1/2B NMDA receptors were co-expressed in Xenopus oocytes and various muscarinic agonists were assessed for their modulatory effects on NMDA receptor-mediated responses. As expected, NMDA receptor-mediated responses were potentiated by oxotremorine-M, oxotremorine or xanomeline when the drugs were applied between subsequent NMDA responses, an effect which was fully blocked by the muscarinic receptor antagonist atropine. However, in oocytes expressing NR1/2B NMDA receptors but not muscarinic M1 receptors, oxotremorine-M co-applied with NMDA also resulted in a potentiation of NMDA currents and this effect was not blocked by atropine, demonstrating that oxotremorine-M is able to directly potentiate NMDA receptors. Oxotremorine, which is a close analogue of oxotremorine-M, and xanomeline, a chemically distinct muscarinic agonist, did not potentiate NMDA receptors by this direct mechanism. Comparing the chemical structures of the three different muscarinic agonists used in this study suggests that the tri-methyl ammonium moiety present in oxotremorine-M is important for the compound's interaction with NMDA receptors.

Grape Seed Proanthocyanidin and Swimming Exercise Protects Against Cognitive Decline: A Study on M1 Acetylcholine Receptors in Aging Male Rat Brain.

Decline in cognition is one of the earliest signs of normal brain aging. Several dietary and non-pharmacological approaches have been tested to slow down this process. The aim of the present study was to assess the influence of grape seed proanthocyanidin extract (GSPE) either individually or in combination with swimming training on acetylcholine esterase activity (AChE) and m1 acetylcholine receptor (m1AChR) on the extent of cognitive decline with aging. The experimental protocol included the oral administration of GSPE (400mg/kg body weight) for 14 weeks to 4 (adult) and 18-month-old (middle-aged) male Wistar rats along with swimming training. They were subjected to behavioral testing followed by biochemical and immunohistochemical analysis. The results demonstrated that GSPE supplementation and swimming training either individually or in combination had an improvement on acquisition and working memory with reduced AChE activity in the medial prefrontal cortex (mPFC) and hippocampus (HC). Immunohistochemical and qRT-PCR evaluation showed an increase in m1AChR protein and mRNA in the CA1 region of HC and also mPFC upon swimming training with GSPE treatment. These beneficial and synergistic effects of GSPE and swimming training are suggestive as interventions in modulating the cognitive function, with GSPE alone being more suitable for middle-aged individuals.

Muscarinic receptor signaling contributes to atypical antipsychotic drug reversal of the phencyclidine-induced deficit in novel object recognition in rats.

Enhancement of cholinergic function via muscarinic acetylcholine receptor M1 agonism improves cognition in some schizophrenia patients. Most atypical antipsychotic drugs, including clozapine and its active metabolite, N-desmethylclozapine, and lurasidone, enhance the release of acetylcholine in key brain regions involved in cognition (e.g. hippocampus). We determined the effect of muscarinic acetylcholine receptor M1 stimulation on novel object recognition and its contribution to the ability of atypical antipsychotic drugs to reverse the novel object recognition deficit in rats withdrawn from subchronic phencyclidine, a rodent model of cognitive impairment in schizophrenia. In control rats, the non-specific muscarinic acetylcholine receptor antagonist, scopolamine, and the M1 selective antagonist, VU0255035, induced a novel object recognition deficit, which was reversed by the M1 agonist, AC260584. Scopolamine fully blocked the effect of clozapine and N-desmethylclozapine, but not lurasidone, to restore novel object recognition in subchronic phencyclidine-treated rats. VU0255035 also blocked these effects of clozapine and N-desmethylclozapine, but not lurasidone; however, the blockade was not as complete as that achieved with scopolamine. Furthermore, subchronic phencyclidine increased hippocampal M1 mRNA expression. These data suggest that M1 agonism is required for clozapine and N-desmethylclozapine to ameliorate the phencyclidine-induced deficit in novel object recognition, additional evidence that M1 agonism is a potential target for treating cognitive impairment in schizophrenia.

Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma.

Asthma often progresses into adulthood from early-life episodes of adverse environmental exposures. However, how the injury to developing lungs contributes to the pathophysiology of persistent asthma remains poorly understood. In this study, we identified an age-related mechanism along the cholinergic nerve-airway smooth muscle (ASM) axis that underlies prolonged airway hyperreactivity (AHR) in mice. We showed that ASM continued to mature until ∼3 wk after birth. Coinciding with postnatal ASM maturation, there was a critical time window for the development of ASM hypercontractility after cholinergic stimulation. We found that allergen exposure in neonatal mice, but not in adult mice, elevated the level and activity of cholinergic nerves (termed neuroplasticity). We demonstrated that cholinergic neuroplasticity is necessary for the induction of persistent AHR after neonatal exposure during rescue assays in mice deficient in neuroplasticity. In addition, early intervention with cholinergic receptor muscarinic (ChRM)-3 blocker reversed the progression of AHR in the neonatal exposure model, whereas β2-adrenoceptor agonists had no such effect. Together, our findings demonstrate a functional relationship between cholinergic neuroplasticity and ASM contractile phenotypes that operates uniquely in early life to induce persistent AHR after allergen exposure. Targeting ChRM3 may have disease-modifying benefits in childhood asthma.-Patel, K. R., Bai, Y., Trieu, K. G., Barrios, J., Ai, X. Targeting acetylcholine receptor M3 prevents the progression of airway hyperreactivity in a mouse model of childhood asthma.

Human Pancreatic Acinar Cells: Proteomic Characterization, Physiologic Responses, and Organellar Disorders in ex Vivo Pancreatitis

Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed exvivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Because data on these human acinar preparations are sparse, we assessed their functional integrity and cellular and organellar morphology using light, fluorescence, and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry. Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid 3-sulfate were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion, and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiological responses to CCh for at least 20 hours. As in rodent acini, human acini exposed to toxic concentrations of CCh and taurolithocholic acid 3-sulfate responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy, and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients, including IL-6, tumor necrosis factor-α, IL-1β, chemokine (C-C motif) ligands 2 and 3, macrophage inhibitory factor, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiological functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.

Melatonin Improves Cognitive Deficits via Restoration of Cholinergic Dysfunction in a Mouse Model of Scopolamine-Induced Amnesia.

Melatonin is known to improve cognitive deficits, and its functions have been studied in various disease models, including Alzheimer's disease. In this study, we investigated effects of melatonin on cognition and the cholinergic system of the septum and hippocampus in a mouse model of scopolamine-induced amnesia. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were administered intraperitoneally to mice for 2 and 4 weeks. The Morris water maze and passive avoidance tests revealed that both treatments of scopolamine significantly impaired spatial learning and memory; however, 2- and 4-week melatonin treatments significantly improved spatial learning and memory. In addition, scopolamine treatments significantly decreased protein levels and immunoreactivities of choline acetyltransferase (ChAT), high-affinity choline transporter (CHT), vesicular acetylcholine transporter (VAChT), and muscarinic acetylcholine receptor M1 (M1R) in the septum and hippocampus. However, the treatments with melatonin resulted in increased ChAT-, CHT-, VAChT-, and M1R-immunoreactivities and their protein levels in the septum and hippocampus. Our results demonstrate that melatonin treatment is effective in improving the cognitive deficits via restoration of the cholinergic system in the septum and hippocampus of a mouse model of scopolamine-induced amnesia.

Functional autoantibodies targeting G protein-coupled receptors in rheumatic diseases.

G protein-coupled receptors (GPCRs) comprise the largest and most diverse family of integral membrane proteins that participate in different physiological processes such as the regulation of the nervous and immune systems. Besides the endogenous ligands of GPCRs, functional autoantibodies are also able to bind GPCRs to trigger or block intracellular signalling pathways, resulting in agonistic or antagonistic effects, respectively. In this Review, the effects of functional GPCR-targeting autoantibodies on the pathogenesis of autoimmune diseases, including rheumatic diseases, are discussed. Autoantibodies targeting β1 and β2 adrenergic receptors, which are expressed by cardiac and airway smooth muscle cells, respectively, have an important role in the development of asthma and cardiovascular diseases. In addition, high levels of autoantibodies against the muscarinic acetylcholine receptor M3 as well as those targeting endothelin receptor type A and type 1 angiotensin II receptor have several implications in the pathogenesis of rheumatic diseases such as Sjögren syndrome and systemic sclerosis. Expanding the knowledge of the pathophysiological roles of autoantibodies against GPCRs will shed light on the biology of these receptors and open avenues for new therapeutic approaches.

Canagliflozin prevents scopolamine-induced memory impairment in rats: Comparison with galantamine hydrobromide action

Canagliflozin (CAN) is a sodium-glucose co-transporter 2 (SGLT2) inhibitor indicated to improve glycemic control in adults with type 2 diabetes mellitus. There is a little information about its effect on the cholinergic system that proposed mechanism for memory improvement occurring by SGLT2 drugs. This study aimed to estimate the effect of CAN as compared to galantamine (GAL) treatments for two weeks on scopolamine hydrobromide (SCO)-induced memory dysfunction in experimental rats. Animals divided into six groups; control (CON), CAN, GAL, SCO, SCO + CAN and SCO + GAL. Results indicated significant decrease in body weights of the CAN groups as compared to control values. Moreover, in the SCO + CAN and SCO + GAL the number of arm entry and number of correct alternation in Y maze task increased and showed improvement in the water maze task, acetylcholinesterase (AChE) activities decreased significantly, while monoamines levels significantly increased compared with the SCO group values. Results also recorded acetylcholine M1 receptor (M1 mAChR) in SCO + CAN or SCO + GAL groups in comparison with the SCO group. The study suggested that canagliflozin might improve memory dysfunction induced by scopolamine hydrobromide via cholinergic and monoamines system.

A novel organotypic 3D sweat gland model with physiological functionality.

Dysregulated human eccrine sweat glands can negatively impact the quality-of-life of people suffering from disorders like hyperhidrosis. Inability of sweating can even result in serious health effects in humans affected by anhidrosis. The underlying mechanisms must be elucidated and a reliable in vitro test system for drug screening must be developed. Here we describe a novel organotypic three-dimensional (3D) sweat gland model made of primary human eccrine sweat gland cells. Initial experiments revealed that eccrine sweat gland cells in a two-dimensional (2D) culture lose typical physiological markers. To resemble the in vivo situation as close as possible, we applied the hanging drop cultivation technology regaining most of the markers when cultured in its natural spherical environment. To compare the organotypic 3D sweat gland model versus human sweat glands in vivo, we compared markers relevant for the eccrine sweat gland using transcriptomic and proteomic analysis. Comparing the marker profile, a high in vitro-in vivo correlation was shown. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), muscarinic acetylcholine receptor M3 (CHRM3), Na+-K+-Cl- cotransporter 1 (NKCC1), calcium-activated chloride channel anoctamin-1 (ANO1/TMEM16A), and aquaporin-5 (AQP5) are found at significant expression levels in the 3D model. Moreover, cholinergic stimulation with acetylcholine or pilocarpine leads to calcium influx monitored in a calcium flux assay. Cholinergic stimulation cannot be achieved with the sweat gland cell line NCL-SG3 used as a sweat gland model system. Our results show clear benefits of the organotypic 3D sweat gland model versus 2D cultures in terms of the expression of essential eccrine sweat gland key regulators and in the physiological response to stimulation. Taken together, this novel organotypic 3D sweat gland model shows a good in vitro-in vivo correlation and is an appropriate alternative for screening of potential bioactives regulating the sweat mechanism.

Alterations of M1 and M4 acetylcholine receptors in the genetically dystonic (dtsz) hamster and moderate antidystonic efficacy of M1 and M4 anticholinergics

Striatal cholinergic dysfunction has been suggested to play a critical role in the pathophysiology of dystonia. In the dtsz hamster, a phenotypic model of paroxysmal dystonia, M1 antagonists exerted moderate antidystonic efficacy after acute systemic administration. In the present study, we examined the effects of the M4 preferring antagonist tropicamid and whether long-term systemic or acute intrastriatal injections of the M1 preferring antagonist trihexyphenidyl are more effective in mutant hamsters. Furthermore, M1 and M4 receptors were analyzed by autoradiography and immunohistochemistry. Tropicamide retarded the onset of dystonic attacks, as previously observed after acute systemic administration of trihexyphenidyl. Combined systemic administration of trihexyphenidyl (30mg/kg) and tropicamide (15mg/kg) reduced the severity in acute trials and delayed the onset of dystonia during long-term treatment. In contrast, acute striatal microinjections of trihexyphenidyl, tropicamid or the positive allosteric M4 receptor modulator VU0152100 did not exert significant effects. Receptor analyses revealed changes of M1 receptors in the dorsomedial striatum, suggesting that the cholinergic system is involved in abnormal striatal plasticity in dtsz hamsters, but the pharmacological data argue against a crucial role on the phenotype in this animal model. However, antidystonic effects of tropicamide after systemic administration point to a novel therapeutic potential of M4 preferring anticholinergics for the treatment of dystonia.

Role of spatial inhomogenity in GPCR dimerisation predicted by receptor association–diffusion models

G protein-coupled receptor (GPCR) association is an emerging paradigm with far reaching implications in the regulation of signalling pathways and therapeutic interventions. Recent super resolution microscopy studies have revealed that receptor dimer steady state exhibits sub-second dynamics. In particular the GPCRs, muscarinic acetylcholine receptor M1 (M1MR) and formyl peptide receptor (FPR), have been demonstrated to exhibit a fast association/dissociation kinetics, independent of ligand binding. In this work, we have developed a spatial kinetic Monte Carlo model to investigate receptor homo-dimerisation at a single receptor resolution. Experimentally measured association/dissociation kinetic parameters and diffusion coefficients were used as inputs to the model. To test the effect of membrane spatial heterogeneity on the simulated steady state, simulations were compared to experimental statistics of dimerisation. In the simplest case the receptors are assumed to be diffusing in a spatially homogeneous environment, while spatial heterogeneity is modelled to result from crowding, membrane micro-domains and cytoskeletal compartmentalisation or ‘corrals’. We show that a simple association–diffusion model is sufficient to reproduce M1MR association statistics, but fails to reproduce FPR statistics despite comparable kinetic constants. A parameter sensitivity analysis is required to reproduce the association statistics of FPR. The model reveals the complex interplay between cytoskeletal components and their influence on receptor association kinetics within the features of the membrane landscape. These results constitute an important step towards understanding the factors modulating GPCR organisation.

MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE.

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-15 MUSCARINIC M3- AND M2- RECEPTOR-ACTIVATED SIGNALING IN THE BLADDER IS INVERSELY REGULATED BY CAVEOLAE. Vivian Cristofaro and Maryrose P. Sullivan Vivian CristofaroVivian Cristofaro More articles by this author and Maryrose P. SullivanMaryrose P. Sullivan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2918AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder smooth muscle (BSM) caveolae are cholesterol-enriched membrane microdomains that augment or attenuate detrusor functional responses by differentially regulating specific receptor-activated signaling pathways. However, BSM contractions induced by cholinergic activation in the rat are not altered by depletion of caveolae, unlike other smooth muscle systems in which muscarinic receptor signaling is evidently mediated by caveolae. Although this discrepancy may reflect the highly specific regulation imparted by caveolae among different tissues and species, a differential regulation of muscarinic M3 and M2 receptor subtypes in the bladder cannot be excluded. This study examined the functional and molecular relationship between caveolae and muscarinic acetylcholine receptor (mAChR) subtypes M3 and M2. METHODS BSM tissue strips were prepared from Sprague Dawley rat bladders after removing the mucosa. Tissue was suspended in organ baths for isometric tension studies. Dose response curves to carbachol (CCh, 1nM-10μM) were generated at baseline, as well as in the presence of 4-DAMP (10nM) or AFDX (0.1μM) to inactive M3 or M2 receptors respectively. Responses to CCh were repeated after incubation with methyl-β-cyclodextrin (mβCD, 15mM), an agent that disrupts caveolae by depleting membrane cholesterol. Interaction between caveolin-1 (Cav-1, a protein required for caveolae biogenesis) and M3 or M2 mAChR subtype were investigated by co-immunoprecipitation. RESULTS Compared to baseline responses, 4-DAMP decreased CCh-induced contractions at each dose. After mβCD treatment and in the presence of 4-DAMP, contractile responses to CCh were significantly enhanced. AFDX had little effect on CCh dose-response curves. However, the subsequent disruption of caveolae in the presence of AFDX attenuated significantly contractions induced by CCh. Immunoreactive bands corresponding to M3 and M2 mAChR subtypes were detected in Cav-1 immunoprecipitates. CONCLUSIONS The opposite effect of mβCD on CCh responses in the presence of M2 or M3 antagonists suggests that caveolae negatively regulate M2- and positively regulate M3-mediated signaling respectively, but this interaction is masked when only the aggregate effect of CCh is examined. Molecular interaction of Cav-1 and mAChRs is consistent with their localization within caveolae. Changes in the balance among caveolin-mAChR interactions, due to loss of caveolae or changes in mAChR subtype expression, may alter responses to cholinergic activation or the efficacy of anti-muscarinic agents in the bladder. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1252 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Vivian Cristofaro More articles by this author Maryrose P. Sullivan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Label-free pharmacological profiling based on dynamic mass redistribution for characterization and authentication of hazardous natural products

Natural products are becoming increasingly popular in multiple fields involving medicines, foods and beverages. However, due to the frequent occurrence of poisoning incidents, their toxicity and safety have caused a serious concern. Here we report a method of biosensor-based two-phase pharmacological profiling (BTPP) for discovery, monitor and control of receptor-targeted natural products. BTPP uses a resonant waveguide grating biosensor for label-free and non-invasive detection of intracellular dynamic mass redistribution (DMR), a phenomenon caused by protein relocalization after receptors receiving stimulation from toxicants. The method can not only facilitate the identification of hazardous materials but also quantify their bioactivity by EC50. As a proof of concept, the method was successfully applied to recognize Daturae Flos (DF), an herb that can antagonize muscarinic acetylcholine M2 receptor and further cause poisoning, from other easily confused species. BTPP combined with high performance liquid chromatography revealed that scopolamine and hyoscyamine in DF were the key marker compounds. Moreover, the method accurately picked out 2 M2 receptor antagonists from 25 natural compounds, displaying its potential in high-throughput screening. This study provides a systematic illustration about the establishment, applicability and advantages of BTPP, which contributes to the safety assessment of natural products in related fields.

A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.

Chemistry-based molecular signature underlying the atypia of clozapine

The central nervous system is functionally organized as a dynamic network of interacting neural circuits that underlies observable behaviors. At higher resolution, these behaviors, or phenotypes, are defined by the activity of a specific set of biomolecules within those circuits. Identification of molecules that govern psychiatric phenotypes is a major challenge. The only organic molecular entities objectively associated with psychiatric phenotypes in humans are drugs that induce psychiatric phenotypes and drugs used for treatment of specific psychiatric conditions. Here, we identified candidate biomolecules contributing to the organic basis for psychosis by deriving an in vivo biomolecule-tissue signature for the atypical pharmacologic action of the antipsychotic drug clozapine. Our novel in silico approach identifies the ensemble of potential drug targets based on the drug’s chemical structure and the region-specific gene expression profile of each target in the central nervous system. We subtracted the signature of the action of clozapine from that of a typical antipsychotic, chlorpromazine. Our results implicate dopamine D4 receptors in the pineal gland and muscarinic acetylcholine M1 (CHRM1) and M3 (CHRM3) receptors in the prefrontal cortex (PFC) as significant and unique to clozapine, whereas serotonin receptors 5-HT2A in the PFC and 5-HT2C in the caudate nucleus were common significant sites of action for both drugs. Our results suggest that D4 and CHRM1 receptor activity in specific tissues may represent underappreciated drug targets to advance the pharmacologic treatment of schizophrenia. These findings may enhance our understanding of the organic basis of psychiatric disorders and help developing effective therapies.