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Study objectives: Narcolepsy type 1 is a rare hypersomnia of central origin, which is caused by loss of hypothalamic neurons that produce the neuropeptides hypocretin-1 and -2. Hypocretin-containing nerve terminals are found in areas known to play a central role in autonomic control and in pain signaling. Cholinergic M2 receptors are found in brain areas involved with the occurrence of hallucinations and cataplexy. In addition to classical symptoms of narcolepsy, the patients suffer frequently from autonomic dysfunction, chronic pain, and hypnagogic/hypnopompic hallucinations. We aimed to test whether narcolepsy type 1 patients have autoantibodies against autonomic beta 2 adrenergic receptor, M2 muscarinic receptors, or nociception receptors. Methods: We tested the serum of ten narcolepsy type 1 patients (five female) for activating beta 2 adrenergic receptor autoantibodies, M2 muscarinic receptor autoantibodies, and nociception receptor autoantibodies. Results: Ten of ten patients were positive for muscarinic M2 receptor autoantibodies (P

The M2 muscarinic acetylcholine receptor (M2R) is a prototypical G protein-coupled receptor (GPCR) that responds to acetylcholine (ACh) and mediates various cellular responses in the nervous system. We recently established Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy for ligand binding to M2R reconstituted in lipid membranes, paving the way to understand the mechanism in atomic detail. However, the obtained difference FTIR spectra upon ligand binding contained ligand, protein, lipid, and water signals, so a vibrational assignment was needed for a thorough understanding. In the present study, we compared difference FTIR spectra between unlabeled and 2-13C labeled ACh, and assigned the bands at 1741 and 1246 cm−1 as the CO and C–O stretches of ACh, respectively. The CO stretch of ACh in M2R is close to that in aqueous solution (1736 cm−1), and much lower in frequency than the free CO stretch (1778–1794 cm−1), indicating a strong hydrogen bond, which probably formed with N4046.52. We propose that a water molecule bridges ACh and N4046.52. The other ACh terminal is positively charged, and it interacts with negatively charged D1033.32. The present study revealed that D1033.32 is deprotonated (negatively charged) in both ACh-bound and free states, a suggested mechanism to stabilize the negative charge of D1033.32 in the free M2R.

Introduction Bronchodilators are the cornerstone of chronic obstructive pulmonary disease (COPD) therapy and long-acting muscarinic antagonists (LAMAs) as a mono or combination treatment play a pivotal role. Several LAMAs are already available on the market in different formulations, but developing a new compound with a higher M3 receptor selectivity and a lower affinity to M2 receptors to increase the therapeutic effect and minimize the adverse effects is still a goal. Moreover, new formulations could improve adherence to therapy. Areas covered This systematic review assesses investigational long-acting muscarinic antagonist in Phase I and II clinical trials over the last decade. It offers insights on whether LAMAs and/or their new formulations in clinical development can become effective treatments for COPD in the future. Expert opinion Research on LAMA seems to have come to a standstill, the few new molecules under study do not show distinctive characteristics compared to the previous ones. Muscarinic antagonist/β2-agonist (MABAs) appear to be the major innovation currently under investigation, and they could theoretically open new research frontiers on the effect between adrenergic and muscarinic interaction in the same cell.

Colonic intramuscular interstitial cells of Cajal (ICC-IM) are associated with cholinergic varicosities, suggesting a role in mediating excitatory neurotransmission. Ca2+ release in ICC-IM activates Ano1, a Ca2+ -activated Cl- conductance, causing tissue depolarization and increased smooth muscle excitability. We employed Ca2+ imaging of colonic ICC-IM in situ, using mice expressing GCaMP6f in ICC to evaluate ICC-IM responses to excitatory neurotransmission. Expression of muscarinic type 2, 3 (M2 , M3 ), and NK1 receptors were enriched in ICC-IM. NK1 receptor agonists had minimal effects on ICC-IM, whereas neostigmine and carbachol increased Ca2+ transients. These effects were reversed by DAU 5884 (M3 receptor antagonist) but not AF-DX 116 (M2 receptor antagonist). Electrical field stimulation (EFS) in the presence of L-NNA and MRS 2500 enhanced ICC-IM Ca2+ transients. Responses were blocked by atropine or DAU 5884, but not AF-DX 116. ICC-IM responses to EFS were ablated by inhibiting Ca2+ stores with cyclopiazonic acid and reduced by inhibiting Ca2+ influx via Orai channels. Contractions induced by EFS were reduced by an Ano1 channel antagonist, abolished by DAU 5884, and unaffected by AF-DX 116. Colonic ICC-IM receive excitatory inputs from cholinergic neurons via M3 receptor activation. Enhancing ICC-IM Ca2+ release and Ano1 activation contributes to excitatory responses of colonic muscles.

Fluorescently labeled dibenzodiazepinone-type muscarinic acetylcholine receptor (MR) antagonists, including dimeric ligands, were prepared using red-emitting cyanine dyes. Probes containing a fluorophore with negative charge showed high M2R affinities (pKi (radioligand competition binding): 9.10-9.59). Binding studies at M1 and M3-M5 receptors indicated a M2R preference. Flow cytometric and high-content imaging saturation and competition binding (M1R, M2R, and M4R) confirmed occupation of the orthosteric site. Confocal microscopy revealed that fluorescence was located mainly at the cell membrane (CHO-hM2R cells). Results from dissociation and saturation binding experiments (M2R) in the presence of allosteric M2R modulators (dissociation: W84, LY2119620, and alcuronium; saturation binding: W84) were consistent with a competitive mode of action between the fluorescent probes and the allosteric ligands. Taken together, these lines of evidence indicate that these ligands are useful fluorescent molecular tools to label the M2R in imaging and binding studies and suggest that they have a dualsteric mode of action.

Regenerative capability of the peripheral nervous system after injury is enhanced by Schwann cells (SCs) producing several growth factors. The clinical use of SCs in nerve regeneration strategies is hindered by the necessity of removing a healthy nerve to obtain the therapeutic cells. Adipose-derived stem cells (ASCs) can be chemically differentiated towards a SC-like phenotype (dASCs), and represent a promising alternative to SCs. Their physiology can be further modulated pharmacologically by targeting receptors for neurotransmitters such as acetylcholine (ACh). In this study, we compare the ability of rat dASCs and native SCs to produce NGF in vitro. We also evaluate the ability of muscarinic receptors, in particular the M2 subtype, to modulate NGF production and maturation from the precursor (proNGF) to the mature (mNGF) form. For the first time, we demonstrate that dASCs produce higher basal levels of proNGF and mature NGF compared to SCs. Moreover, muscarinic receptor activation, and in particular M2 subtype stimulation, modulates NGF production and maturation in both SCs and dASCs. Indeed, both cell types express both proNGF A and B isoforms, as well as mNGF. After M2 receptor stimulation, proNGF-B (25 kDa), which is involved in apoptotic processes, is strongly reduced at transcript and protein level. Thus, we demonstrate that dASCs possess a stronger neurotrophic potential compared to SCs. ACh, via M2 muscarinic receptors, contributes to the modulation and maturation of NGF, improving the regenerative properties of dASCs.

Binding of muscarinic ligands, both antagonists and agonists, and their effects on the conformation of the M2 acetylcholine receptor were modeled in silico and compared to experimental data. After docking of antagonists to the M2 receptor in an inactive conformation (3UON, 5ZK3, 5ZKB, or 5ZKB) and agonists in an active conformation (4MQS), 100 ns of conventional molecular dynamics (MD) followed by 500 ns of accelerated MD was run. Conventional MD revealed ligand-specific interactions with the receptor. Antagonists stabilized the receptor in an inactive conformation during accelerated MD. The receptor in complex with various agonists attained different conformations specific to individual agonists. The magnitude of the TM6 movement correlated with agonist efficacy at the non-preferential Gs pathway. The shape of the intracellular opening where the receptor interacts with a G-protein was different for the classical agonist carbachol, super-agonist iperoxo, and Gi/o-biased partial agonists JR-6 and JR-7, being compatible with experimentally observed agonist bias at the G-protein level. Moreover, a wash-resistant binding of the unique agonist xanomeline associated with interactions with membrane lipids was formed during accelerated MD. Thus, accelerated MD is suitable for modeling of ligand-specific receptor binding and receptor conformations that is essential for the design of experiments aimed at identification of the secondary binding sites and understanding molecular mechanisms underlying receptor activation.

Synthetic cathinones (SCs) are designer, psychostimulant drugs of abuse that primarily act on monoamine transporters; little is known about their off-target liability. Abuse of pyrrolidine-containi...

A series of fluorescent dibenzodiazepinone-type muscarinic acetylcholine M(2) receptor (M(2)R) ligands was synthesized using various fluorescent dyes (5-TAMRA, λ(ex)/λ(em) ≈ 547/576 nm; BODIPY 630/650, λ(ex)/λ(em) ≈ 625/640 nm; pyridinium dye Py-1, λ(ex)/λ(em) ≈ 611/665 nm and pyridinium dye Py-5, λ(ex)/λ(em) ≈ 465/732 nm). All fluorescent probes exhibited high M(2)R affinity (pK(i) (radioligand competition binding): 8.75-9.62, pK(d) (flow cytometry): 8.36–9.19), a very low preference for the M(2)R over the M(1) and M(4) receptors and moderate to pronounced M(2)R selectivity compared to the M(3) and M(5) receptors. The presented fluorescent ligands are considered useful molecular tools for future studies using methods such as fluorescence anisotropy and BRET based MR binding assays.

Background To compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS). Methods Male C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed. Results Pirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level. Conclusions The administration of pirenzepine and atropine may have beneficial effects on septic shock.

There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of timescales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the τ-Random Acceleration Molecular Dynamics (τRAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the β2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.TABLE OF CONTENTS GRAPHIC

Obesity-related asthma often presents with more severe symptoms than non-obesity-related asthma and responds poorly to current treatments. Both insulin resistance and hyperinsulinemia are common in obesity. We have shown that increased insulin mediates airway hyperreactivity in diet-induced obese rats by causing neuronal M2 muscarinic receptor dysfunction, which normally inhibits acetylcholine release from parasympathetic nerves. Decreasing insulin with streptozotocin prevented airway hyperreactivity and M2 receptor dysfunction. The objective of the present study was to investigate whether pioglitazone, a hypoglycemic drug, prevents airway hyperreactivity and M2 receptor dysfunction in obese rats. Male rats fed a low- or high-fat diet were treated with pioglitazone or PBS by daily gavage. Body weight, body fat, fasting insulin, and bronchoconstriction and bradycardia in response to electrical stimulation of vagus nerves and to aerosolized methacholine were recorded. Pilocarpine, a muscarinic receptor agonist, was used to measure M2 receptor function. Rats on a high-fat diet had potentiated airway responsiveness to vagal stimulation and dysfunctional neuronal M2 receptors, whereas airway responsiveness to methacholine was unaffected. Pioglitazone reduced fasting insulin and prevented airway hyperresponsiveness and M2 receptor dysfunction but did not change inflammatory cytokine mRNA expression in alveolar macrophages. High-fat diet, with and without pioglitazone, had tissue-specific effects on insulin receptor mRNA expression. In conclusion, pioglitazone prevents vagally mediated airway hyperreactivity and protects neuronal M2 muscarinic receptor function in obese rats.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of seven transmembrane receptors that mediates a wide variety of extracellular signals. We show here, for the first time in a GPCR, that the carboxyl terminal fragment of the muscarinic M2receptor, containing the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site located in the third intracellular loop. Single cell imaging reveals that M2tail, whose expression is upregulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here it controls oxygen consumption, cell proliferation and the formation of reactive oxygen species by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human induced pluripotent stem cells, reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signalling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism thar cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

AZD8871 is a novel muscarinic antagonist and β2-adrenoceptor agonist in development for chronic obstructive pulmonary disease. This study describes the pharmacological profile of AZD8871 in in vitro and in vivo assays. AZD8871 is potent at the human M3 receptor (pIC50 in binding assays: 9.5) and shows kinetic selectivity for the M3 (half-life: 4.97 hours) over the M2 receptor (half-life: 0.46 hour). It is selective for the β2-adrenoceptor over the β1 and β3 subtypes (3- and 6-fold, respectively) and shows dual antimuscarinic and β2-adrenoceptor functional activity in isolated guinea pig tissue (pIC50 in electrically stimulated trachea: 8.6; pEC50 in spontaneous tone isolated trachea: 8.8, respectively), which are sustained over time. AZD8871 exhibits a higher muscarinic component than batefenterol in human bronchi, with a shift in potency under propranolol blockade of 2- and 6-fold, respectively, together with a persisting relaxation (5.3% recovery at 8 hours). Nebulized AZD8871 prevents acetylcholine-induced bronchoconstriction in both guinea pig and dog with minimal effects on salivation and heart rate at doses with bronchoprotective activity. Moreover, AZD8871 shows long-lasting effects in dog, with a bronchoprotective half-life longer than 24 hours. In conclusion, these studies demonstrate that AZD8871 is a dual-acting molecule with a high muscarinic component and a long residence time at the M3 receptor; moreover, its preclinical profile in animal models suggests a once-daily dosing in humans and a favorable safety profile. Thus, AZD8871 has the potential to be a next generation of inhaled bronchodilators in respiratory diseases.

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Our previous investigations have revealed that microinjection of acetylcholine (ACh) into the cerebellar fastigial nucleus (FN) induces a systemic blood pressure (BP) depressor response and that the muscarinic ACh (mACh) receptor contributes substantially to this process. This study was determined to explore the role of muscarinic ACh receptor-2 (M2 receptor) in ACh-mediated BP regulation and its underlying electrophysiological mechanism. ACh mixed with methoctramine hydrate (MCT, a selective M2 receptor antagonist) and normal saline (0.9% NaCl) were separately microinjected into the FN of anaesthetized rats, and the mean arterial pressure during the reactive time (the duration for BP to return to its basal level) was calculated; the responses of cholinergic reagents to the inward currents in FN neurons were also examined via patch-clamp recording in vitro; in addition, the receptor expression in the FN was confirmed by immunohistochemistry. The results demonstrated that MCT effectively blocked the ACh-mediated BP depressor response and the ACh-induced inward currents in FN neurons; moreover, M2 receptors were unequivocally distributed in the FN. Taken together, the results indicate that M2 receptors in the FN may be involved in ACh-mediated BP down-regulation by tuning neuronal excitation.

Azithromycin (AZM) has been used for the treatment of asthma and chronic obstructive pulmonary disease (COPD); however, the effects and underlying mechanisms of AZM remain largely unknown. The effects of AZM on airway smooth muscles (ASMs) and the underlying mechanisms were studied using isometric muscle force measurements, the examination of lung slices, imaging, and patch-clamp techniques. AZM completely inhibited acetylcholine (ACH)-induced precontraction of ASMs in animals (mice, guinea pigs, and rabbits) and humans. Two other macrolide antibiotics, roxithromycin and Klaricid, displayed a decreased inhibitory activity, and the aminoglycoside antibiotics penicillin and streptomycin did not have an inhibitory effect. Precontractions were partially inhibited by nifedipine (selective inhibitor of L-type voltage-dependent Ca2+ channels (LVDCCs)), Pyr3 (selective inhibitor of TRPC3 and/or STIM/Orai channels, which are nonselective cation channels (NSCCs)), and Y-27632 (selective inhibitor of Rho-associated kinase (ROCK)). Moreover, LVDCC- and NSCC-mediated currents were inhibited by AZM, and the latter were suppressed by the muscarinic (M) 2 receptor inhibitor methoctramine. AZM inhibited LVDCC Ca2+ permeant ion channels, M2 receptors, and TRPC3 and/or STIM/Orai, which decreased cytosolic Ca2+ concentrations and led to muscle relaxation. This relaxation was also enhanced by the inhibition of Ca2+ sensitization. Therefore, AZM has potential as a novel and potent bronchodilator. The findings of this study improve the understanding of the effects of AZM on asthma and COPD.

A previous body of work in bovine and rodent models shows that cholinergic agonists modulate the secretion of steroid hormones from the adrenal cortex. In this study we used live-cell Ca2+ imaging to investigate cholinergic activity in the HAC15 human adrenocortical carcinoma cell line. The cholinergic agonists carbachol and acetylcholine triggered heterogeneous Ca2+ oscillations that were strongly inhibited by antagonists with high affinity for the M3 muscarinic receptor subtype, while preferential block of M1 or M2 receptors was less effective. Acute exposure to carbachol and acetylcholine modestly elevated aldosterone secretion in HAC15 cells, and this effect was also diminished by M3 inhibition. HAC15 cells expressed relatively high levels of mRNA for M3 and M2 receptors, while M1 and M5 mRNA were much lower. In conclusion, our data extend previous findings in non-human systems to implicate the M3 receptor as the dominant muscarinic receptor in the human adrenal cortex.

Neuromodulation by acetylcholine plays a vital role in shaping the physiology and functions of cerebral cortex. Cholinergic neuromodulation influences brain-state transitions, controls the gating of cortical sensory stimulus responses, and has been shown to influence the generation and maintenance of persistent activity in prefrontal cortex. Here we review our current understanding of the role of muscarinic cholinergic receptors in primate prefrontal cortex during its engagement in the performance of working memory tasks. We summarize the localization of muscarinic receptors in prefrontal cortex, review the effects of muscarinic neuromodulation on arousal, working memory and cognitive control tasks, and describe the effects of muscarinic M1 receptor stimulation and blockade on the generation and maintenance of persistent activity of prefrontal neurons encoding working memory representations. Recent studies describing the pharmacological effects of M1 receptors on prefrontal persistent activity demonstrate the heterogeneity of muscarinic actions and delineate unexpected modulatory effects discovered in primate prefrontal cortex when compared with studies in rodents. Understanding the underlying mechanisms by which muscarinic receptors regulate prefrontal cognitive control circuitry will inform the search of muscarinic-based therapeutic targets in the treatment of neuropsychiatric disorders.

Bioluminescence resonance energy transfer (BRET ) is a very sensitive technique employed to study protein-protein interactions, including G-protein-coupled receptor (GPCR ) hetero- and homo-dimerization. Recently, BRET has also been used to investigate the interaction between GPCRs (e.g.: α2 adrenergic receptor, muscarinic M2 receptor, dopaminergic D2 receptor) and nonvisual arrestins. Within the last decade an increasing interest arose toward opioid agonists with limited activation of arrestin-dependent signaling pathways, as they are believed to be effective analgesics with reduced adverse effects. Here a BRET protocol is described to investigate interactions between the kappa opioid receptor (KOR ) and nonvisual arrestins (arrestin-2 and arrestin-3) in HEK-293 cells, both under basal conditions and after exposure to KOR ligands.

There is a growing appreciation of the importance of drug-target binding kinetics for lead optimization. For G protein-coupled receptors (GPCRs), which mediate signaling over a wide range of time scales, the drug dissociation rate is often a better predictor of in vivo efficacy than binding affinity, although it is more challenging to compute. Here, we assess the ability of the τ-Random Acceleration Molecular Dynamics (τRAMD) approach to reproduce relative residence times and reveal dissociation mechanisms and the effects of allosteric modulation for two important membrane-embedded drug targets: the β2-adrenergic receptor and the muscarinic acetylcholine receptor M2. The dissociation mechanisms observed in the relatively short RAMD simulations (in which molecular dynamics (MD) simulations are performed using an additional force with an adaptively assigned random orientation applied to the ligand) are in general agreement with much more computationally intensive conventional MD and metadynamics simulations. Remarkably, although decreasing the magnitude of the random force generally reduces the number of egress routes observed, the ranking of ligands by dissociation rate is hardly affected and agrees well with experiment. The simulations also reproduce changes in residence time due to allosteric modulation and reveal associated changes in ligand dissociation pathways.

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.

Muscarinic Receptor Classification, M. Caulfield Muscarinic Acetylcholine Receptor Signaling Pathways in Smooth Muscle, R.A.J. Challiss and J.L. Blank Muscarinic Receptors and Gastrointestinal Smooth Muscle, F.J. Ehlert, E.A. Thomas, E.H. Gerstin, and M.T. Griffin Muscarinic Receptors and Genitourinary Smooth Muscle, R.M. Eglen and S.S. Hegde Prejunctional Muscarinic Receptors and Smooth Muscle, N. Watson Index

The biological effects resulting from parasympathetic nerve stimulation in genitourinary tissues are mediated chiefly through muscarinic cholinoceptors of which there are five known subtypes (M1-M5). With the exception of the corpus cavernosum, which relaxes in response to muscarinic receptor agonism, most genitourinary tissues contract, either directly or indirectly, when exposed to muscarinic receptor agonists. In recent years, the availability of selective pharmacological tools has allowed us to make considerable strides in our understanding of the functional role of individual muscarinic receptor subtype(s) in genitourinary tissues. The muscarinic receptor subtype mediating the contractile response is dependent on the species, tissue, and in some cases, region of tissue, under study. Recent studies have unmasked the functional role of M2 receptors in the urinary bladder and these findings may set the stage for the development of safer and more efficacious subtype selective muscarinic antagonists for the treatment of overactive bladder. Unambiguous elucidation of muscarinic receptor pharmacology in the urethra, prostate, vas deferens and corpus cavernosum requires additional rigorous studies. Lastly, the identity of the muscarinic receptor mediating uterine contraction continues to generate controversy.

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a downregulation in the expression of ATP “binding cassette” G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors. Fil: Español, Alejandro Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Salem, Agustina Reina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Di Bari, María. Università degli Studi di Roma "La Sapienza"; Italia Fil: Cristofaro, Ilaria. Università degli Studi di Roma "La Sapienza"; Italia Fil: Sanchez, Yamila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina Fil: Tata, Ada Maria. Università degli Studi di Roma "La Sapienza"; Italia Fil: Sales, María Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; Argentina

Atropine is commonly used to counter the effects of the parasympathetic neurotransmitter acetylcholine on heart rate in clinical practice, such as in the perioperative period; however, individual differences in the response to atropine are huge. The association between SCN10A/voltage-gated sodium channel 1.8 (NaV1.8) and cardiac conduction has been demonstrated; however, the exact role of SCN10A/NaV1.8 in the heart rate response to atropine remains unclear. To identify the role of SCN10A variants that influence the heart rate responses to atropine, we carried out a retrospective study in 1,005 Han Chinese subjects. Our results showed that rs6795970 was associated with the heart rate response to atropine. The heart rate responses to atropine and methoctramine in NaV1.8 knockout mice were lower, whereas the heart rate response to isoproterenol was like those in wild type mice. Furthermore, we observed that the NaV1.8 blocker A-803467 alleviated the heart rate response to atropine in wild type mice. The retrospective study revealed a previously unknown role of NaV1.8 in controlling the heart rate response to atropine, as shown by the animal study, a speculative mechanism that may involve the cardiac muscarinic acetylcholine receptor M2.

Optimal attention performance requires cholinergic modulation of corticothalamic neurons in the prefrontal cortex. These pyramidal cells express specialized nicotinic acetylcholine receptors containing the α5 subunit encoded by Chrna5. Disruption of this gene impairs attention, but the advantage α5 confers for the detection of endogenous cholinergic signaling is unknown. To ascertain this underlying mechanism, we used optogenetics to stimulate cholinergic afferents in prefrontal cortex brain slices from compound-transgenic wild-type and Chrna5 knockout mice of both sexes. These electrophysiological experiments identify that Chrna5 is critical for the rapid onset of the postsynaptic cholinergic response. Loss of α5 slows cholinergic excitation and delays its peak, and these effects are observed in two different optogenetic mouse lines. Disruption of Chrna5 does not otherwise perturb the magnitude of the response, which remains strongly mediated by nicotinic receptors and tightly controlled by autoinhibition via muscarinic M2 receptors. However, when conditions are altered to promote sustained cholinergic receptor stimulation, it becomes evident that α5 also works to protect nicotinic responses against desensitization. Rescuing Chrna5 disruption thus presents the double challenge of improving the onset of cholinergic signaling without triggering desensitization. Here, we identify that an agonist for the unorthodox α-α nicotinic binding site can allosterically enhance this cholinergic pathway considered vital for attention. Minimal NS9283 treatment restores the rapid onset of the postsynaptic cholinergic response without triggering desensitization. Taken together, this work demonstrates the advantages of speed and resilience that Chrna5 confers on endogenous cholinergic signaling, defining a critical window of interest for cue detection and attentional processing.Significance statementThe α5 nicotinic receptor subunit (Chrna5) is important for attention, but its advantage in detecting endogenous cholinergic signals is unknown. Here, we show that α5 subunits permit rapid cholinergic responses in prefrontal cortex and protect these responses from desensitization. Our findings clarify why Chrna5 is required for optimal attentional performance under demanding conditions. To treat the deficit arising from Chrna5 disruption without triggering desensitization, we enhanced nicotinic receptor affinity using NS9283 stimulation at the unorthodox α-α nicotinic binding site. This approach successfully restored the rapid-onset kinetics of endogenous cholinergic neurotransmission. In summary, we reveal a previously unknown role of Chrna5 as well as an effective approach to compensate for genetic disruption and permit fast cholinergic excitation of prefrontal attention circuits.

Experimental and clinical studies of cardiac pathology associated with epilepsy have demonstrated an impact on the autonomic nervous system (ANS). However, the underlying molecular mechanism has not been fully elucidated. Molecular investigation of the neurotransmitters related receptor and ion channel directing ANS might help in understanding the associated mechanism. In this paper, we investigated the role of acetylcholine (ACh), which demonstrates both sympathetic and parasympathetic roles in targeted expression in terms of the relevant receptor and ion channel. Inwardly rectifying potassium (Kir) channels play a significant role in maintaining the resting membrane potential and controlling cell excitability and are prominently expressed in both the excitable and non-excitable tissues. The immunoreactivity of ACh-activated Kir3.1 channel and muscarinic ACh receptors (M2) in autonomic centers such as the brainstem, vagus nerve (VN) and atria of heart was confirmed by both histological staining and pathological tissue analysis. Significant upregulations of Kir3.1 and M2 receptors were observed in pentylenetetrazol (PTZ)-kindled epileptic rats for all related tissues investigated, whereas no pathological difference was observed. These findings provide proof-of-concept that changes in ACh-associated immunoreactivity might be linked to the ANS dysfunctions associated with epilepsy.

The aim of the present study was to characterize the specific binding sites for [N-methyl-3H]-scopolamine ([3H]-NMS), a radioligand for labeling muscarinic acetylcholine receptors (mAChRs), in membranes of four heart chambers obtained from adult male British United Turkey (BUT) Big 6 (“meat-type”) and Crollwitzer (“wild-type”) turkeys. MAChR subtypes were examined by inhibiting [3H]-NMS binding with subtype selective non-labelled receptor antagonists. In all left and right atria as well as left and right ventricles of both turkey breeds, the specific [3H]-NMS binding was saturable, reversible and of high affinity (KD range: 0.5–1.0 nM). The maximum receptor density (Bmax) was not significantly different between the four cardiac chambers of BUT Big 6 turkeys, but a significant difference was found between atria and ventricles of Crollwitzer turkeys. Moreover, significant lower Bmax was found in the atria of Crollwitzer turkeys than in the atria of BUT Big 6, while the ventricular Bmax was significantly higher. In all cardiac chambers, unlabeled mAChR antagonists competed for specific [3H]-NMS binding sites in a concentration-dependent manner, suggesting the presence of the M3 and M2 receptor subtypes, whereby the latter was the predominant subtype. The presence of the M1 subtype could not be excluded. In conclusion, there was a difference between BUT Big 6 (“meat-type”) and Crollwitzer (“wild-type”) turkeys with regard to receptor density in heart chambers with dominant M2 and M3 receptor subtypes.

F Nascimento was supported by The Alfred Dunhill Links Foundation. G B Miles and M J Broadhead received support from Biotechnology and Biological Sciences Research Council Grant BB/M021793/1. L Zagoraiou and E Tsape were supported by Fondation Santé. Spinal motor networks are formed by diverse populations of interneurons that set the strength and rhythmicity of behaviors such as locomotion. A small cluster of cholinergic interneurons, expressing the transcription factor Pitx2, modulates the intensity of muscle activation via ‘C-bouton’ inputs to motoneurons. However, the synaptic mechanisms underlying this neuromodulation remain unclear. Here, we confirm in mice that Pitx2+ interneurons are active during fictive locomotion and that their chemogenetic inhibition reduces the amplitude of motor output. Furthermore, after genetic ablation of cholinergic Pitx2+ interneurons, M2 receptor-dependent regulation of the intensity of locomotor output is lost. Conversely, chemogenetic stimulation of Pitx2+ interneurons leads to activation of M2 receptors on motoneurons, regulation of Kv2.1 channels and greater motoneuron output due to an increase in the inter-spike afterhyperpolarization and a reduction in spike half-width. Our findings elucidate synaptic mechanisms by which cholinergic spinal interneurons modulate the final common pathway for motor output. Publisher PDF

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M(2) receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1‐(thiophen‐2‐ylmethyl)‐3,6‐dihydro‐2H‐pyridinium and 1‐methyl‐1‐(thiophen‐2‐ylmethyl)‐3,6‐dihydro‐2H‐pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M(2) muscarinic receptors and displaying bias towards the G(i) signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist‐specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non‐steroidal and non‐opioid analgesics acting via M(2) and M(4) muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an “ingredient-gene-path” network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P<0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P<0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P<0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

PLOS ONE 15(1), e0226954 (2020). doi:10.1371/journal.pone.0226954, Published by PLOS, San Francisco, California, US

In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100 μM) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.

Uncertainty over the mechanism of signaling via G protein-coupled receptors (GPCRs) relates in part to questions regarding their supramolecular structure. GPCRs and heterotrimeric G proteins are known to couple as monomers under various conditions. Many GPCRs form oligomers under many of the same conditions, however, and the biological role of those complexes is unclear. We have used dual-color fluorescence correlation spectroscopy to identify oligomers of the M2 muscarinic receptor and of Gi1 in purified preparations and live Chinese hamster ovary cells. Measurements on differently tagged receptors (i.e., eGFP-M2 and mCherry-M2) and G proteins (i.e., eGFP-Gαi1β1γ2 and mCherry-Gαi1β1γ2) detected significant cross-correlations between the two fluorophores in each case, both in detergent micelles and in live cells, indicating that both the receptor and Gi1 can exist as homo-oligomers. Oligomerization of differently tagged Gi1 decreased upon the activation of co-expressed wild-type M2 receptor by an agonist. Measurements on a tagged M2 receptor (M2-mCherry) and eGFP-Gαi1β1γ2 co-expressed in live cells detected cross-correlations only in the presence of an agonist, which therefore promoted coupling of the receptor and the G protein. The effect of the agonist was retained when a fluorophore-tagged receptor lacking the orthosteric site (i.e., M2(D103A)-mCherry) was co-expressed with the wild-type receptor and eGFP-Gαi1β1γ2, indicating that the ligand acted via an oligomeric receptor. Our results point to a model in which an agonist promotes transient coupling of otherwise independent oligomers of the M2 receptor on the one hand and of Gi1 on the other and that an activated complex leads to a reduction in the oligomeric size of the G protein. They suggest that GPCR-mediated signaling proceeds, at least in part, via oligomers.

Qiong Wu,1,2,* Yu Cao,1,* Meixia Liu,1 Fang Liu,1 Adelheid H Brantner,3 Yang Yang,1 Yun Wei,1 Yu Zhou,4 Zhiyong Wang,1 Lina Ma,1 Feixue Wang,1 Hui Pei,1 Hao Li1 1Department of Geriatrics, Xiyuan Hospital, China Academy of Chinese Medical Sciences, Beijing 100091, People’s Republic of China; 2Department of Ophthalmology, Dongfang Hospital, Beijing University of Chinese Medicine, Beijing 100078, People’s Republic of China; 3Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria; 4Department of Polymer Chemistry, Zernike Institute for Advanced Materials, Groningen 9747 AG, The Netherlands*These authors contributed equally to this workCorrespondence: Hao Li; Hui PeiDepartment of Geriatrics, Xiyuan Hospital of China Academy of Chinese Medical Sciences, 1 Xiyuan Caochang Road, Haidian District, Beijing 100091, People’s Republic of ChinaTel +86 10 6283 5631Email xyhplihao1965@126.com; phxydoctor@126.comPurpose: Clinical trials have illustrated that Shenmayizhi decoction (SMYZ) could improve the cognitive functions in patients with dementia. However, the mechanism needs to be explored.Methods: Fifty adult male rats (Wistar strain) were divided into five groups equally and randomly, including control, model, and SMYZ of low dose, medium dose and high dose. Rats in each group received a daily gavage of respective treatment. Rats in control and model group were administrated by the same volume of distilled water. Memory impairment was induced by intraperitoneal administration of scopolamine (0.7 mg/kg) for 5 continuous days. Four weeks later, Morris water maze (MWM) was performed to evaluate the spatial memory in all rats. Then, rats were sacrificed and the hippocampus was removed for further tests. Furthermore, Western blot analysis was employed to assess the levels of acetylcholine M1 receptor (M1), acetylcholine M2 receptor (M2), acetylcholinesterase (AChE) and cholineacetyltransferase (ChAT). AChE and ChAT activities were determined.Results: The SMYZ decoction significantly improved behavioral performance of rats in high dose. The SMYZ decoction in three doses exhibited anti-acetylcholinesterase activity. In addition, a high dose of SMYZ promoted ChAT activity. Moreover, a high dose of SMYZ increased the level of ChAT and declined the level of AChE assessed by Western blotting. Besides, an increased level of M1 receptor was found after treatment.Conclusion: Shenmayizhi decoction could mitigate scopolamine-induced cognitive deficits through the preventative effect on cholinergic system dysfunction.Keywords: dementia, Shenmayizhi decoction, scopolamine, acetylcholine M1 receptor, acetylcholinesterase, cholineacetyltransferase &nbsp

Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6–8.8) in an atropine-sensitive manner (apparent pA2 8.6–8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5–6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 μM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1 μM, yielding a biphasic curve with a pIC50 of 8.6 for the falling part and a pEC50 of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10 μM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory Gs protein comes into play at high concentrations of a muscarinic receptor agonist.

Although 7,8-dihydroxyflavone (7,8-DHF), a synthetic agonist specific for tyrosine kinase receptor B (TrkB), has been reported to promote intestinal dynamics, its effect on gastric dynamics has not been studied as yet. In this study, we explored how 7,8-DHF affected the carbachol (CCh)-induced contraction of rat gastric muscle by way of measuring the contractile tension of muscular strips. We found that although 7,8-DHF did not directly cause contraction of gastric muscle, it enhanced CCh-induced, instead of substance P- or high K+ -induced, contraction. The enhancing role of 7,8-DHF was partially blocked by ANA-12, a blocker specific for TrkB the activation of which in the gastric strips was evidenced by its phosphorylation. Although 7,8-DHF alone did not activate : phospholipase C (PLC)-γ in gastric muscle, CCh did, and importantly, the combined treatment with CCh + 7,8-DHF activated more PLC-γ. U73122, an antagonist to PLC-γ blocked both the CCh-induced and the 7,8-DHF-enhanced/CCh-induced contraction by ~30%. To pursue how 7,8-DHF could augment CCh-activated PLC-γ phosphorylation, we first examined the effect of 7,8-DHF on the expression of muscarinic receptors in gastric muscle and found that 7,8-DHF specifically increased M3 but not M2 receptor expression possibly through TrkB/Akt (protein kinase B) pathway because the Akt antagonist, LY294002 significantly suppressed the 7,8-DHF-augmemted M3 expression and completely blocked the 7,8-DHF-enhanced cholinergic contraction. Supporting the result, Akt phosphorylation in the gastric muscle was enhanced by 7,8-DHF treatment. The in vivo experiment showed that orally fed 7,8-DHF increased gastric emptying rate. The results imply a possibility that 7,8-DHF may be developed into a drug in the future for enhancing gastric dynamics.

The hippocampus, a brain region that is important for spatial navigation and episodic memory, benefits from a rich diversity of neuronal cell-types. Through the use of an intersectional genetic viral vector approach in mice, we report novel hippocampal neurons which we refer to as LINCs, as they are long-range inhibitory neuronal nitric oxide synthase (nNOS)-expressing cells. LINCs project to several extrahippocampal regions including the tenia tecta, diagonal band, and retromammillary nucleus, but also broadly target local CA1 cells. LINCs are thus both interneurons and projection neurons. LINCs display regular spiking non-pyramidal firing patterns, are primarily located in the stratum oriens or pyramidale, have sparsely spiny dendrites, and do not typically express somatostatin, VIP, or the muscarinic acetylcholine receptor M2. We further demonstrate that LINCs can strongly influence hippocampal function and oscillations, including interregional coherence. The identification and characterization of these novel cells advances our basic understanding of both hippocampal circuitry and neuronal diversity.

Muscarinic acetylcholine receptors have been suggested to be implicated in arginine–vasopressin secretion because intracerebroventricular muscarinic agonist administration induces arginine–vasopressin release into the circulation. Although which subtype is involved in the regulation of arginine–vasopressin secretion is unclear, M2 receptors have been reported to be highly expressed in the hypothalamus. In the present study, M2 receptor-knockout mice were used to elucidate whether M2 receptor regulates arginine–vasopressin synthesis in the paraventricular nuclei and supraoptic nuclei of the hypothalamus. The number of arginine–vasopressin-immunoreactive neurons in M2 receptor-knockout mice was significantly decreased in the supraoptic nuclei, but not in the paraventricular nuclei compared with wild-type mice. Plasma arginine–vasopressin level in M2 receptor-knockout mice was also significantly lower than in the wild-type mice. Urinary volume and frequency as well as water intake in M2 receptor-knockout mice were significantly higher than those in wild-type mice. The V2 vasopressin receptor expression in kidneys of M2 receptor-knockout mice was comparable with that of wild-type mice, and increased urination in M2 receptor-knockout mice was significantly decreased by administration of desmopressin, a specific V2 receptor agonist, suggesting that V2 receptors in the kidneys of M2 receptor-knockout mice are intact. These results suggest that M2 receptors promote arginine–vasopressin synthesis in the supraoptic nuclei and play a role in the regulation and maintenance of body fluid.

The muscarinic M2 acetylcholine receptor, one of the few G-protein coupled receptors that has not only been crystallized in both active and inactive conformations but also in the presence of a positive allosteric modulator, is an interesting system to study the molecular mechanisms of GPCR activation and ligand allosterism. Here, we have employed molecular dynamics (MD) simulations (adding to 14 μs in total) to study conformational changes triggered by the inverse agonist R-(−)-3-quinuclidinyl-benzilate (QNB) in the structure of the active M2 receptor (PBD ID 4MQS) after replacement of the agonist iperoxo by the inverse agonist QNB. This permitted us to identify the sequence of events in the deactivation mechanism of the M2 acetylcholine receptor, which results first in the rearrangement of the transmission switch, the subsequent opening of the extracellular portion of the receptor and finally, the closure of the intracellular part. We also evaluate the effect of the positive allosteric modulator LY211962...

Aims Rho/Rho-kinase (ROCK) signaling has extensively been shown to take part in mammalian smooth muscle contractions in response to diverse agents yet its role in the contraction of amphibian smooth muscle has not been investigated. Therefore, we aimed to explore any role of this pathway in the contractions of frog stomach smooth. Main methods The strips were prepared and suspended in organ baths filled with Ringer solution. Changes in the circular strips of the frog stomach muscle length were recorded isotonically with a force transducer in organ baths. Key findings Carbachol (CCh) exerted both phasic and tonic contractions. In contrast, atropin abolished all types of contractions by CCh. The phasic contractions were suppressed by a Ca2+ channel blocker, nifedipine but not by the ROCK inhibitor, Y-27632. However, the tonic contractions were markedly attenuated by Y-27632. Selective M1 receptor blocker, pirenzepin, selective M3 receptor blocker and DAMP had no effects on CCh-elicited contractions. On the other hand, selective M2 receptor blocker, AF-DX suppressed all types of contractile activity by CCh. Significance These data suggest that M2 receptor activation could mainly mediate CCh-induced phasic and tonic contractions, and ROCK seems to be involved in the CCh-induced tonic but not phasic contractions of the frog stomach smooth muscle.

The effect of sarin on the binding parameters (KD & Bmax) of M2 muscarinic acetylcholine receptor (mAChR) was studied 24h and 1 week post exposure. Male & female Sprague-Daweley rats were poisoned with 1XLD50 sarin (80μg/kg, im) followed by treatment of trimedoxime bromide and atropine (7.5:5mg/kg, im) 1min later. Brains were removed and analyzed for M2 mAChR binding, using [3H]AFDX384, an M2 selective antagonist. A significant increase in KD of M2 mAChR was found in the cortex 24h post poisoning, displaying elevation from 4.65±1.16 to 8.45±1.06nM and 5.24±0.93 to 9.29±1.56nM in male and female rats, respectively. A rise in KD was also noted 1 week following exposure from 5.04±1.20 to 11.75±2.78 and from 5.37±1.02 to 11.66±1.73nM, presenting an added increase of 51 and 40% (compared to 24h) in males and females, respectively. Analysis of M2 receptor density (Bmax) revealed a significant reduction of 68% in males and insignificant reduction of 22% in females, 24h after sarin exposure which was followed by 37% recovery in males and 100% recovery in females, 1 week later. These results indicate that sarin induces a long-term decreased affinity in M2 mAChR (elevated KDs) and a transient effect on the number of this receptor subtype (Bmax). We hypothesize that the reduced affinity of the M2 receptors (negative auto-regulatory receptors) may cause long-term brain deficits by impairing the normal regulation release of ACh into the synaptic cleft.

Objective To predict the main active ingredients, potential targets and molecular mechanisms of Yuan Zhi powder in treatment of dementia by using network pharmacology.Methods A database of chemical constituents of Yuan Zhi powder was constructed by using databases and literatures. Potential targets were predicted by reverse molecular docking, and then a component-target network was constructed. The target network of Alzheimer’s disease (AD) was mapped and analyzed to obtain the “active ingredient-AD target” network. The key targets were screened through network analysis. Finally, the rationality of the prediction was analyzed by comparing with the target reported in the literatures.Results There were 180 chemical constituents acting on the AD target, and the targets included three key targets (cyclooxygenase-2, muscarinic acetylcholine receptor M1, and muscarinic acetylcholine receptor M2) and an important target (acetylcholine esterase). Alzheimer’s disease may be treated by regulating the activity of acetylcholine receptors and the binding to β-amyloid protein, and its biological process may be concentrated in the acetylcholine receptor signal pathway, negative regulation of synaptic transmission and so on.Conclusion The fact that Yuan Zhi powder can treat AD is consistent with the characteristics of multi-components-multi-targets-multiple pathways of traditional Chinese medicine. The important targets obtained from network analysis have a large proportion in literature research, so network analysis have some rationality. Keywords: Yuan Zhi powder, Network pharmacology, Active ingredients–AD targets network, Network analysis, Dementia

Muscarinic acetylcholine receptors (mAChR) are known to be related to learning and memory processes. Inactivation of mAChR by cholinergic antagonists have been shown to produce amnesia in a variety of behavioral tasks. In this study, we investigated the role of M1 and M2 AChR on passive avoidance learning and plasticity of synapses formed by Schaffer collaterals in freely moving rats. Experiments were performed using Wistar male rats. Seven days before testing, a recording electrode was lowered in the CA1 region under chloral hydrate anaesthesia to record the field excitatory postsynaptic potential (fEPSP) in response to Schaffer collateral stimulation. Selective M2 receptor antagonists methoctramine and selective M1 receptors antagonist pirenzepine were intraperitoneally injected immediately after training. The effects on memory retention were examined using passive avoidance training. We measured latency of the first entry into a dark compartment of the chamber. fEPSP amplitude and slope ratio were measured before shock presentation, 90 min after the shock, and 24 hour after the shock. Methoctramine significantly impaired behavior in the passive avoidance test but pirenzepine did not induce any changes compared to control. Our results showed that pirenzepine but not methoctramine supressed the amplitude of fEPSPs. On the other hand, intracerebroventricular methoctramine administration impaired passive avoidance learning and increased the amplitude of fEPSP.

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.

The muscarinic M2 receptor (M2R) has been shown to display voltage-sensitive agonist binding, based on G protein-activated inward rectifier potassium channel (GIRK) opening and radioligand binding at different membrane voltages. A conserved aspartate in transmembrane segment (TM) II of M2R, D69, has been proposed as the voltage sensor. While a recent paper instead presented evidence of tyrosines in TMs III, VI, and VII acting as voltage sensors, these authors were not able to record GIRK channel activation by a D69N mutant M2R. In the present study, we succeeded in recording ACh-induced GIRK channel activation by this mutant at −80 and 0 mV. The acetylcholine EC50 was about 2.5-fold higher at 0 mV, a potency shift very similar to that observed at wild-type M2R, indicating that voltage sensitivity persists at the D69N mutant. Thus, our present observations corroborate the notion that D69 is not responsible for voltage sensitivity of the M2R.