Berberine inhibits intracellular Ca2+ signals in mouse pancreatic acinar cells through M3 muscarinic receptors: Novel target, mechanism, and implication.

Berberine, a natural isoquinoline alkaloid, exhibits a variety of pharmacological effects, but the pharmacological targets and mechanisms remain elusive. Here, we report a novel finding that berberine inhibits acetylcholine (ACh)-induced intracellular Ca2+ oscillations, mediated through an inhibition of the muscarinic subtype 3 (M3) receptor. Patch-clamp recordings and confocal Ca2+ imaging were applied to acute dissociated pancreatic acinar cells prepared from CD1 mice to examine the effects of berberine on ACh-induced Ca2+ oscillations. Whole-cell patch-clamp recordings showed that berberine (from 0.1 to 10 µM) reduced ACh-induced Ca2+ oscillations in a concentration-dependent manner, and this inhibition also depended on ACh concentrations. The inhibitory effect of berberine neither occurred in intracellular targets nor extracellular cholecystokinin (CCK) receptors, chloride (Cl-) channels, and store-operated Ca2+ channels. Together, the results demonstrate that berberine directly inhibits the muscarinic M3 receptors, further confirmed by evidence of the interaction between berberine and M3 receptors in pancreatic acinar cells.

Cevimeline co-treatment attenuates olanzapine-induced metabolic disorders via modulating hepatic M3 muscarinic receptor: AMPKα signalling pathway in female rats.

BACKGROUND: Olanzapine is one of the most commonly used antipsychotic drugs; however, its metabolic disorders are the main obstacle in the clinic. Olanzapine is a potent antagonist of the M3 acetylcholine muscarinic receptor (M3R), while the downregulated hepatic M3R-AMPKα signalling pathway is involved in metabolic disorders. AIM: This study investigated the effects of chronic co-treatment with cevimeline (an agonist of M3Rs) in attenuating olanzapine-induced metabolic disorders and the underlying mechanisms. METHODS: Forty-eight adult female Sprague-Dawley rats were treated orally with olanzapine (2 mg/kg, 3 times/day (t.i.d.)) and/or cevimeline (9 mg/kg, t.i.d.), or control (vehicle) for 9 weeks. RESULTS: Cevimeline co-treatment significantly attenuated olanzapine-induced body weight gain and glucolipid metabolic disorders. Importantly, cevimeline co-treatment attenuated olanzapine-induced upregulation of M3Rs, while the co-treatment improved olanzapine-induced downregulation of AMPKα in the liver. Cevimeline co-treatment attenuated olanzapine-induced dyslipidaemia by modulating the hepatic M3R-AMPKα downstream pathways. Cevimeline co-treatment also improved lower activated AKT-GSK3ß signalling to reverse impairment of glucose metabolism and insulin resistance caused by chronic olanzapine treatment. CONCLUSION: These results not only support the important role of M3R antagonism and its related AMPKα and downstream pathways in antipsychotic-induced metabolic disorders but also indicate that these pathways might be promising targets for pharmacological intervention to control these side effects caused by antipsychotic therapy.

Discovery of M3 Antagonist-PDE4 Inhibitor Dual Pharmacology Molecules for the Treatment of Chronic Obstructive Pulmonary Disease.

In this paper, we report the discovery of dual M3 antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled treatment of pulmonary diseases. The identification of dual compounds was enabled by the intuition that the fusion of a PDE4 scaffold derived from our CHF-6001 series with a muscarinic scaffold through a common linking ring could generate compounds active versus both the transmembrane M3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M3 nanomolar affinity coupled with nanomolar PDE4 inhibition, which translated into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.

Effects of (a Combination of) the Beta2-Adrenoceptor Agonist Indacaterol and the Muscarinic Receptor Antagonist Glycopyrrolate on Intrapulmonary Airway Constriction.

Expression of bronchodilatory ß2-adrenoceptors and bronchoconstrictive muscarinic M3-receptors alter with airway size. In COPD, (a combination of) ß2-agonists and muscarinic M3-antagonists (anticholinergics) are used as bronchodilators. We studied whether differential receptor expression in large and small airways affects the response to ß2-agonists and anticholinergics in COPD. Bronchoprotection by indacaterol (ß2-agonist) and glycopyrrolate (anticholinergic) against methacholine- and EFS-induced constrictions of large and small airways was measured in guinea pig and human lung slices using video-assisted microscopy. In guinea pig lung slices, glycopyrrolate (1, 3 and 10 nM) concentration-dependently protected against methacholine- and EFS-induced constrictions, with no differences between large and small intrapulmonary airways. Indacaterol (0.01, 0.1, 1 and 10 µM) also provided concentration-dependent protection, which was greater in large airways against methacholine and in small airways against EFS. Indacaterol (10 µM) and glycopyrrolate (10 nM) normalized small airway hyperresponsiveness in COPD lung slices. Synergy of low indacaterol (10 nM) and glycopyrrolate (1 nM) concentrations was greater in LPS-challenged guinea pigs (COPD model) compared to saline-challenged controls. In conclusion, glycopyrrolate similarly protects large and small airways, whereas the protective effect of indacaterol in the small, but not the large, airways depends on the contractile stimulus used. Moreover, findings in a guinea pig model indicate that the synergistic bronchoprotective effect of indacaterol and glycopyrrolate is enhanced in COPD.

Discovery of a novel class of inhaled dual pharmacology muscarinic antagonist and ß2 agonist (MABA) for the treatment of chronic obstructive pulmonary disease (COPD).

The targeting of both the muscarinic and ß-adrenergic pathways is a well validated therapeutic approach for the treatment of chronic obstructive pulmonary disease (COPD). In this communication we report our effort to incorporate two pharmacologies into a single chemical entity, whose characteristic must be suitable for a once daily inhaled administration. Contextually, we aimed at a locally acting therapy with limited systemic absorption to minimize side effects. Our lung-tailored design of bifunctional compounds that combine the muscarinic and ß-adrenergic pharmacologies by the elaboration of the muscarinic inhibitor 7, successfully led to the potent, pharmacologically balanced muscarinic antagonist and ß2 agonist (MABA) 13.

Synthesis and anticancer evaluation of 6-azacyclonol-2,4,6-trimethylpyridin-3-ol derivatives: M3 muscarinic acetylcholine receptor-mediated anticancer activity of a cyclohexyl derivative in androgen-refractory prostate cancer.

We recently reported 2,4,5-trimethylpyridin-3-ol with C(6)-azacyclonol, whose code name is BJ-1207, showing a promising anticancer activity by inhibiting NOX-derived ROS in A549 human lung cancer cells. The present study was focused on structural modification of the azacyclonol moiety of BJ-1207 to find a compound with better anticancer activity. Ten new compounds (3A-3J) were prepared and evaluated their inhibitory actions against proliferation of eighteen cancer cell lines as a primary screening. Among the ten derivatives of BJ-1207, the effects of compounds 3A and 3J on DU145 and PC-3, androgen-refractory cancer cell lines (ARPC), were greater than the parent compound, and compound 3A showed better activity than 3J. Antitumor activity of compound 3A was also observed in DU145-xenografted chorioallantoic membrane (CAM) tumor model. In addition, the ligand-based target prediction and molecular docking study using DeepZema® server showed compound 3A was a ligand to M3 muscarinic acetylcholine receptor (M3R) which is overexpressed in ARPC. Carbachol, a muscarinic receptor agonist, concentration dependently increased proliferation of DU145 in the absence of serum, and it also activated NADPH oxidase (NOX). The carbachol-induced proliferation and NOX activity was significantly blocked by compounds 3A in a concentration-dependent manner. This finding might become a new milestone in the development of pyridinol-based anti-cancer agents against ARPC.

Kinase and GPCR polypharmacological approach for the identification of efficient anticancer medicines.

Discovery of an anticancer medicine using a single target protein has often been unsuccessful due to the complexity of pathogenic mechanisms as well as the presence of redundant signaling pathways. In this work, we attempted to find promising anticancer drug candidates by simultaneously targeting casein kinase 1 delta (CK1δ) and muscarinic acetylcholine receptor M3 (M3R). Through the structure-based virtual screening and de novo design with the modified potential function for protein-ligand binding, a series of benzo[4,5]imidazo[1,2-a][1,3,5]triazine-2-amine (BITA) derivatives were identified as CK1δ inhibitors and also as M3R antagonists. The biochemical potencies of these bifunctional molecules reached the nanomolar and low-micromolar levels with respect to CK1δ and M3R, respectively. A common interaction feature in the calculated CK1δ-inhibitor and M3R-antagonist complexes is that the BITA moiety is well-stabilized in the orthosteric site of M3R and the hinge region of CK1δ through the establishment of the three hydrogen bonds and the hydrophobic contacts in the vicinity. The computational and experimental results found in this work exemplify the efficiency of kinase and GPCR polypharmacology in developing anticancer medicines.

Autoantibodies Blocking M3 Muscarinic Receptors Cause Postganglionic Cholinergic Dysautonomia.

A 10-year-old girl presented with ileus, urinary retention, dry mouth, lack of tears, fixed dilated pupils, and diffuse anhidrosis 7 days after a febrile illness. We hypothesized that her syndrome was due to autoimmunity against muscarinic acetylcholine receptors, blocking their activation. Using an indirect enzyme-linked immunosorbent assay for all 5 muscarinic receptors (M1 -M5 ), we identified in the patient's serum antibodies that selectively bound to M3 receptors. In vitro functional studies confirmed that these autoantibodies selectively blocked M3 receptor activation. Thus, autoantibodies against M3 acetylcholine receptors cause acute postganglionic cholinergic dysautonomia. ANN NEUROL 2020;88:1237-1243.

M3 but not M4 muscarinic receptors in the rostromedial tegmental nucleus are involved in the acquisition of morphine-induced conditioned place preference.

Opioids strongly inhibit GABAergic neurons in the rostromedial tegmental nucleus (RMTg) that expresses µ-opioid receptors to induce rewarding and psychomotor effects. M3 and M4 muscarinic receptors are co-localized with µ-opioid receptors at these GABAergic neurons. This study explored whether RMTg M3 and M4 muscarinic receptors are involved in regulating opioid-induced reward and locomotion via a conditioned place preference (CPP) paradigm. Selective muscarinic receptor agonists and antagonists were both singly and combinatorically injected into the RMTg to examine their effects on the acquisition of systemic morphine-induced CPP and locomotor activity. The M3 muscarinic receptor agonist, pilocarpine, inhibited the acquisition of morphine-induced CPP, whereas its antagonist, 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP, 1 µg/side), reversed the inhibitory effect of pilocarpine (30 µg/side). Additionally, 4-DAMP increased locomotor activity while pilocarpine (30 µg/side) partially decreased locomotor activity when combined with morphine. In contrast, the M4 muscarinic receptor agonist, LY2033298 (0.1 and 0.2 µg/side), and antagonist, tropicamide (20 and 40 µM/side), did not affect the acquisition of morphine-induced CPP or locomotor activity. Taken together, our findings suggest that RMTg M3 muscarinic receptors are involved in opioid-induced rewarding and psychomotor effects. Therefore, RMTg M3 muscarinic receptors may represent a promising target for the treatment of opioid addiction.

Novel Potent Muscarinic Receptor Antagonists: Investigation on the Nature of Lipophilic Substituents in the 5- and/or 6-Positions of the 1,4-Dioxane Nucleus.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M1-M5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b, with a cis configuration between the CH2N+(CH3)3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed pKi values for mAChRs higher than those of 2 and a selectivity profile analogous to that of the clinically approved drug oxybutynin. The study of the enantiomers of 3b and the corresponding tertiary amine 33b revealed that the eutomers are (2S,6S)-(-)-3b and (2S,6S)-(-)-33b, respectively. Docking simulations on the M3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M3/M2 selectivity, which might limit cardiovascular side effects, make 3b a valuable starting point for the design of novel antagonists potentially useful in peripheral diseases in which M3 receptors are involved.

Regiospecific Introduction of Halogens on the 2-Aminobiphenyl Subunit Leading to Highly Potent and Selective M3 Muscarinic Acetylcholine Receptor Antagonists and Weak Inverse Agonists.

Muscarinic M3 receptor antagonists and inverse agonists displaying high affinity and subtype selectivity over the antitarget M2 are valuable pharmacological tools and may enable improved treatment of chronic obstructive pulmonary disease (COPD), asthma, or urinary incontinence. On the basis of known M3 antagonists comprising a piperidine or quinuclidine unit attached to a biphenyl carbamate, 5-fluoro substitution was responsible for M3 subtype selectivity over M2, while 3'-chloro substitution substantially increased affinity through a σ-hole interaction. Resultantly, two piperidinyl- and two quinuclidinium-substituted biphenyl carbamates OFH243 (13n), OFH244 (13m), OFH3911 (14n), and OFH3912 (14m) were discovered, which display two-digit picomolar affinities with Ki values from 0.069 to 0.084 nM, as well as high selectivity over the M2 subtype (46- to 68-fold). While weak inverse agonistic properties were determined for the biphenyl carbamates 13m and 13n, neutral antagonism was observed for 14m and 14n and tiotropium under identical assay conditions.

The dosage-dependent effects of cevimeline in preventing olanzapine-induced metabolic side-effects in female rats.

Olanzapine has been used for the treatment of schizophrenia and other mental disorders. However, it is associated with serious weight gain and other metabolic side-effects. The antagonistic affinity of olanzapine to muscarinic M3 receptors has been evidenced as one of the main contributors for its weight gain and other metabolic side-effects. Therefore, this study investigated whether the co-treatment of cevimeline (a M3 receptor agonist) could prevent the metabolic side-effects associated with olanzapine medication. Female Sprague Dawley rats were treated orally with olanzapine (2 mg/kg, t.i.d.) and/or cevimeline at 3 dosages (3, 6, 9 mg/kg, t.i.d.), or vehicle for two weeks. Weight gain and food/water intake were measured throughout the drug treatment period. Intraperitoneal glucose tolerance tests and open field tests were conducted. Olanzapine-treated rats demonstrated significantly elevated body weight gain, food intake, feeding efficiency, total white fat mass, liver mass, and plasma triglyceride levels, which could be partly reversed by the co-treatment with cevimeline in a dosage-dependent manner. In general, the body weight gain can only be reversed by the co-treatment of 9 mg/kg cevimeline. The cevimeline co-treatment decreased plasma triglyceride and glucose levels compared with olanzapine only treatment. The results suggested a dosage-dependent effect of cevimeline in ameliorating olanzapine-induced weight gain and metabolic side-effects, which supports further clinical trials using cevimeline to control weight gain and metabolic side-effects caused by antipsychotic medications.

A review of the pharmacokinetics of M3 muscarinic receptor antagonists used for the treatment of asthma.

Introduction: There is solid evidence that in patients with poorly controlled severe asthma despite the use of ICS and LABA, the addition of LAMAs, such as tiotropium, significantly increases the time to the first severe exacerbation and provides a modest but sustained bronchodilation. However, only a very limited number of pharmacokinetic studies with these agents have been performed in asthmatic patients.Areas covered: The pharmacokinetic profile of inhaled tiotropium, umeclidinium and glycopyrronium in healthy volunteers and that of inhaled tiotropium and umeclidinium in asthmatic patients have been reviewed.Expert opinion: In asthmatic patients, LAMAs are rapidly absorbed into the systemic compartment and demonstrate bi-exponential elimination (rapidly declining plasma concentrations followed by slow apparent terminal elimination). Apparently, the severity of asthma does not change the pharmacokinetics of LAMAs. The limited information available is focused on the plasma pharmacokinetic profile of these drugs and, consequently, although suitable for establishing a systemic safety profile, it does not tell us much about possible therapeutic efficacy of LAMAs in asthmatics because quantification of systemic plasma values is neither at the airways, which are their site of action nor representative of their transport to this site.

The Traditional Medicine Banhasasim-Tang Depolarizes Pacemaker Potentials of Cultured Interstitial Cells of Cajal through M3 Muscarinic and 5-HT3 Receptors in Murine Small Intestine.

BACKGROUND: Banhasasim-tang (BHSST) is a classic herbal formulation in traditional Chinese medicine widely used for gastrointestinal (GI) tract motility disorder. We investigated the effects of BHSST on the pacemaker potentials of cultured interstitial cells of Cajal (ICCs) in small intestine in vitro and its effects on GI motor functions in vivo. METHODS: We isolated ICCs from the small intestines and recorded pacemaker potentials in cultured ICCs with the whole-cell patch-clamp configuration in vitro. Intestinal transit rates (ITR%) were investigated in normal mice and GI motility dysfunction (GMD) mouse models in vivo. RESULTS: BHSST (20-50 mg/mL) depolarized pacemaker potentials and decreased their amplitudes in a concentration-dependent manner. Pretreatment with methoctramine (a muscarinic M2 receptor antagonist) did not inhibit BHSST-induced pacemaker potential depolarization. However, when we applied 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; a muscarinic M3 receptor antagonist), BHSST-induced effects were blocked. Pretreatment with Y25130 (a 5-HT3 receptor antagonist) blocked BHSST-induced effects in ICCs. In addition, when we applied 4-DAMP and Y25130 together, BHSST-induced effects were completely blocked. Pretreatment with Ca2+-free solution or thapsigargin inhibited BHSST-induced effects. Moreover, BHSST blocked both the transient receptor potential melastatin (TRPM) 7 and voltage-sensitive calcium-activated chloride (anoctamin-1, ANO1) channels. In normal mice, ITR% values were significantly increased by BHSST in a dose-dependent manner. The ITR% of GMD mice was significantly reduced relative to those of normal mice, which were significantly reversed by BHSST in a dose-dependent manner. CONCLUSION: These results suggested that BHSST depolarizes the pacemaker potentials of ICCs in a dose-dependent manner through the M3 and 5-HT3 receptors via internal and external Ca2+-dependent and TRPM7- and ANO1-independent pathways in vitro. Moreover, BHSST increased ITR% in vivo in normal mice and GMD mouse models. Taken together, the results of this study showed that BHSST had the potential for development as a prokinetic agent in GI motility function.

Central muscarinic receptor subtypes (M1 and M3) involved in carbacol-induced hypophagia in neonatal broiler chicken.

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist), 4-DAMP (M3 receptor antagonist), and PD102807 (M4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD3) neonatal broiler chicken.Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3-5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection.Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group (P < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake (P < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol (P < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake (P < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol (P > 0.05).Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors in neonatal chicken.

Rewarding effects of M4 but not M3 muscarinic cholinergic receptor antagonism in the rostromedial tegmental nucleus.

The rostromedial tegmental nucleus (RMTg) receives inputs from the laterodorsal tegmental and pedunculopontine tegmental nuclei, the two principle brainstem cholinergic nuclei. We tested the effects of RMTg M3 and M4 muscarinic cholinergic receptor antagonism in a conditioned place preference (CPP) paradigm in mice. RMTg infusions of the M3 muscarinic cholinergic receptor antagonist 1,1-Dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) do not result in the acquisition of CPP but increase locomotor activation. By contrast, RMTg infusions of the M4 muscarinic cholinergic receptor antagonist Tropicamide result in the acquisition of CPP but do not increase locomotor activation. The rewarding effects of RMTg Tropicamide infusions are dopamine-dependent as systemic pre-treatment with the broad-spectrum dopamine receptor antagonist flupenthixol prevents the acquisition of CPP induced by RMTg Tropicamide infusions. Under conditions of systemic dopamine receptor blockade, RMTg Tropicamide infusions significantly increase locomotor activation. These data provide further support for an important role of endogenous cholinergic input to the RMTg in reward function and suggest that the contributions of RMTg cholinergic input to rewarding and locomotor-activating effects involve differential contributions of RMTg M4 and M3 muscarinic receptors, respectively.

A Selective M1 and M3 Receptor Antagonist, Penehyclidine Hydrochloride, Exerts Antidepressant-Like Effect in Mice.

Recent studies indicate that anti-muscarinic receptor is a prospective strategy to treat depression. Although non-selective antagonist of muscarinic receptor scopolamine exhibits rapid and robust antidepressant-like effect, it still has various side effects including abuse risk. Penehyclidine hydrochloride (PHC) is a novel clinical anti-cholinergic drug derived from scopolamine in China, which selectively blocks M1 and M3 muscarinic receptor. Therefore, the objective of this study was to evaluate whether PHC would manifest antidepressant-like effects. Forced swim test (FST), tail suspension test (TST) and chronic unpredictable mild stress (CUMS) model of depression were explored to assess the antidepressant-like effect. Western blotting was further performed to detect the effects of PHC on the brain-derived neurotrophic factor (BDNF) signal cascade. Immunofluorescence was used to observe the activation of astrocyte. Moreover, different pharmacological inhibitors were applied to clarify the antidepressant-like mechanism. The results of the present experiments revealed that PHC decreased the immobility time of FST and TST in mice. In the CUMS model, PHC rapidly ameliorated anhedonia-like behavior (within 4 days), accompanying with the enhanced expression of BDNF and phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) in the hippocampus. In addition, blockade of the BDNF release by verapamil and activation of its Trk B receptor by K252a, rather than inhibition of opioid system by naloxone or sigma receptor by BD1047, abolished the antidepressant-like effects of PHC in mice. The findings suggest that PHC, an anti-muscarinic drug in clinical use, elicits rapid onset antidepressant-like effect, shedding light on the development of new antidepressants.

Balanced cholinergic modulation of spinal locomotor circuits via M2 and M3 muscarinic receptors.

Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.

R2-8018 reduces the proliferation and migration of non-small cell lung cancer cells by disturbing transactivation between M3R and EGFR.

AIMS: The M3 muscarinic acetylcholine receptor (M3R) is a G protein-coupled receptor that is expressed in cases of non-small cell lung cancer (NSCLC). Previous studies demonstrated that M3R antagonists reduce the proliferation of NSCLC. However, how antagonists inhibit the NSCLC proliferation and migration is still little known. This study aims to investigate the mechanism of M3R involved in the growth of NSCLC. MAIN METHODS: The CRISPR/Cas9 was used to knock out (KO) the M3R gene. A real-time cell analyzer (RTCA) was used to record the proliferation of NSCLC cells. The migration and cell cycle of NSCLC cells were evaluated with scratch test and flow cytometry (FCM), respectively. Antibody microarray analysis was performed to detect the expression of proteins after antagonizing M3R and knocking out of M3R, subsequently some of these important proteins were verified by western blot. KEY FINDINGS: The proliferation and migration of NSCLC cells were inhibited by M3R antagonist R2-8018 and knocking out of M3R. Antagonism or knocking out of M3R reduced the phosphorylation of EGFR. Moreover, c-Src and ß-arrestin-1 are involved in the mechanism of how the inhibition of M3R affects EGFR in NSCLC. Further study demonstrated that PI3K/AKT and MEK/ERK signal pathways are involved in M3R-induced EGFR transactivation in NSCLC, and the molecules involved in the cell cycle progression and migration of NSCLC cells were identified. SIGNIFICANCE: This further understanding of the relationship between M3R and NSCLC facilitates the design of therapeutic strategy with M3R antagonist as an adjuvant drug for NSCLC treatment.

Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate, a muscarinic receptor antagonist with anti-inflammatory activity.

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(-)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1 and 5, (R)-(-)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(-)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(-)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.