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Nicotinic receptors (NRs) play an important role in the cholinergic regulation of heart functions, and converging evidence suggests a diverse repertoire of NR subunits in the heart. A recent hypothesis about the plasticity of β NR subunits suggests that β2-subunits and β4-subunits may substitute for each other. In our study, we assessed the hypothetical β-subunit interchangeability in the heart at the level of mRNA. Using two mutant mice strains lacking β2 or β4 NR subunits, we examined the relative expression of NR subunits and other key cholinergic molecules. We investigated the physiology of isolated hearts perfused by Langendorff's method at basal conditions and after cholinergic and/or adrenergic stimulation. Lack of β2 NR subunit was accompanied with decreased relative expression of β4-subunits and α3-subunits. No other cholinergic changes were observed at the level of mRNA, except for increased M3 and decreased M4 muscarinic receptors. Isolated hearts lacking β2 NR subunit showed different dynamics in heart rate response to indirect cholinergic stimulation. In hearts lacking β4 NR subunit, increased levels of β2-subunits were observed together with decreased mRNA for acetylcholine-synthetizing enzyme and M1 and M4 muscarinic receptors. Changes in the expression levels in β4 -/- hearts were associated with increased basal heart rate and impaired response to a high dose of acetylcholine upon adrenergic stimulation. In support of the proposed plasticity of cardiac NRs, our results confirmed subunit-dependent compensatory changes to missing cardiac NRs subunits with consequences on isolated heart physiology. NEW & NOTEWORTHY In the present study, we observed an increase in mRNA levels of the β2 NR subunit in β4 -/- hearts but not vice versa, thus supporting the hypothesis of β NR subunit plasticity that depends on the specific type of missing β-subunit. This was accompanied with specific cholinergic adaptations. Nevertheless, isolated hearts of β4 -/- mice showed increased basal heart rate and a higher sensitivity to a high dose of acetylcholine upon adrenergic stimulation.

Context affects the salience and visibility of image elements in visual scenes. Collinear flankers can enhance or decrease the perceptual and neuronal sensitivity to flanked stimuli. These effects are mediated through lateral interactions between neurons in the primary visual cortex (area V1), in conjunction with feedback from higher visual areas. The strength of lateral interactions is affected by cholinergic neuromodulation. Blockade of muscarinic receptors should increase the strength of lateral intracortical interactions, while nicotinic blockade should reduce thalamocortical feed-forward drive. Here we test this proposal through local iontophoretic application of the muscarinic receptor antagonist scopolamine and the nicotinic receptor antagonist mecamylamine, while recording single cells in parafoveal representations in awake fixating macaque V1. Collinear flankers generally reduced neuronal contrast sensitivity. Muscarinic and nicotinic receptor blockade equally reduced neuronal contrast sensitivity. Contrary to our hypothesis, flanker interactions were not systematically affected by either receptor blockade.

Arecoline is one of the nicotinic acid-based alkaloids, which is found in the betel nut. In addition to its function as a muscarinic agonist, arecoline exhibits several adverse effects, such as inducing growth retardation and causing developmental defects in animal embryos, including zebrafish, chicken, and mice. In this study, we aimed to study the potential adverse effects of waterborne arecoline exposure on zebrafish larvae locomotor activity and investigate the possible mechanism of the arecoline effects in zebrafish behavior. The zebrafish behavior analysis, together with molecular docking and the antagonist co-exposure experiment using muscarinic acetylcholine receptor antagonists were conducted. Zebrafish larvae aged 96 h post-fertilization (hpf) were exposed to different concentrations (0.001, 0.01, 0.1, and 1 ppm) of arecoline for 30 min and 24 h, respectively, to find out the effect of arecoline in different time exposures. Locomotor activities were measured and quantified at 120 hpf. The results showed that arecoline caused zebrafish larvae locomotor hyperactivities, even at a very low concentration. For the mechanistic study, we conducted a structure-based molecular docking simulation and antagonist co-exposure experiment to explore the potential interactions between arecoline and eight subtypes, namely, M1a, M2a, M2b, M3a, M3b, M4a, M5a, and M5b, of zebrafish endogenous muscarinic acetylcholine receptors (mAChRs). Arecoline was predicted to show a strong binding affinity to most of the subtypes. We also discovered that the locomotion hyperactivity phenotypes triggered by arecoline could be rescued by co-incubating it with M1 to M4 mAChR antagonists. Taken together, by a pharmacological approach, we demonstrated that arecoline functions as a highly potent hyperactivity-stimulating compound in zebrafish that is mediated by multiple muscarinic acetylcholine receptors.

Although s ynaptic transmission in motor pathways can be regulated by neuromodulators, such as acetylcholine, few studies have examined how cholinergic activity affects cortical and spinal motor circuits following muscle contractions of varying intensities. This was a human, double-blinded, placebo-controlled, crossover study. Participants attended two sessions where they were administered either a placebo or 25 mg of promethazine. Electromyography of the abductor digiti minimi (ADM) was measured for all conditions. Motor evoked potentials (MEPs) were obtained via motor cortical transcranial magnetic stimulation (TMS), and F waves were obtained via ulnar nerve electrical stimulation. MEPs and F waves were examined: 1 ) when the muscle was at rest; 2 ) after the muscle had been active; and 3 ) after the muscle had been fatigued. MEPs were unaffected by muscarinic receptor blockade when measurements were recorded from resting muscle or following a 50% isometric maximal voluntary contraction (MVC). However, muscarinic receptor blockade increased MEP area following a 10-s MVC ( P = 0.019) and following a fatiguing 60-s MVC ( P = 0.040). F wave area and persistence were not affected by promethazine for any muscle contraction condition. Corticospinal excitability was influenced by cholinergic effects when voluntary drive to the muscle was high. Given that spinal motoneurone excitability remained unaffected, it is likely that cholinergic effects are influential within the motor cortex during strong muscle contractions. Future research should evaluate how cholinergic effects alter the relationship between subcortical structures and the motor cortex, as well as brainstem neuromodulatory pathways and spinal motoneurons. NEW & NOTEWORTHY The relationship between motor function and cholinergic circuitry in the central nervous system is complex. Although many studies have approached this issue at the cellular level, few studies have examined cholinergic mechanisms in humans performing muscle contractions. This study demonstrates that blockade of muscarinic acetylcholine receptors enhances motor evoked potentials (elicited with transcranial magnetic stimulation) following strong muscle contractions, but not weak muscle contractions.

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca 2+ -channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca 2+ -chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K + -buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K + -stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.

The family of human muscarinic acetylcholine receptors (MRs) is characterized by a high sequence homology among the five subtypes (M 1 R-M 5 R), being the reason for a lack of subtype selective MR ligands. In continuation of our work on dualsteric dibenzodiazepinone-type M 2 R antagonists, a series of M 2 R ligands containing a dibenzodiazepinone pharmacophore linked to small basic peptides was synthesized (64 compounds). The linker moiety was varied with respect to length, number of basic nitrogens (0-2) and flexibility. Besides proteinogenic basic amino acids (Lys, Arg), shorter homologues of Lys and Arg, containing three and two methylene groups, respectively, as well as D-configured amino acids were incorporated. The type of linker had a marked impact on M 2 R affinity and also effected M 2 R selectivity. In contrast, the structure of the basic peptide rather determined M 2 R selectivity than M 2 R affinity. For example, the most M 2 R selective compound (UR-CG188, 89) with picomolar M 2 R affinity (pK i 9.60), exhibited a higher M 2 R selectivity (ratio of K i M 1 R/M 2 R/M 3 R/M 4 R/M 5 R: 110:1:5200:55:2300) compared to the vast majority of reported M 2 R preferring MR ligands. For selected ligands, M 2 R antagonism was confirmed in a M 2 R miniG protein recruitment assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Introduction: The β 2 adrenergic receptor (β 2 AR) is a member of G protein-coupled receptors (GPCRs) that mediate the majority of cellular responses to external stimuli. The agonists can cause smooth muscle relaxation; therefore, many β 2 AR agonists have been developed especially for the treatment of pulmonary disorders such as asthma and chronic obstructive pulmonary disease (COPD). Many new natural and synthetic compounds have been discovered and developed as novel β 2 AR agonists over the past 5 years., Areas Covered: This review offers an update for the development of β 2 AR agonists in the patents published from 2015 to 2020, including new natural and synthetic compounds for the treatment of asthma and COPD. In particular, the latest patents about compounds possessing both muscarinic receptor antagonist and β 2 adrenergic receptor agonist activity are reviewed., Expert Opinion: β 2 AR agonists have been developed extensively for the treatment of asthma and COPD. In the past 5 years, novel agonists from both natural sources and synthetic methods were intensively developed. Compounds possessing both muscarinic receptor antagonist and β 2 AR agonist activity represent a new trend in this area because they are possibly able to act together in a synergistic fashion, therefore, relieve the symptoms of patients through two distinct mechanisms.

Background: Phosalone (Pln) is an organophosphorus pesticide acetylcholinesterase (AChE) inhibitor. Blockade of AChE amplifies ACh signaling that is related to depressive symptoms. The effects of Pln exposure were evaluated on depressive behavior in mice and the involvement of muscarinic ACh receptor (MAChR) was assessed., Material and Methods: After measuring total activity in the locomotor test the immobility time during the forced swimming test (FST) in male mice was evaluated as an index of depression. Pln single dose was administered by gavage feeding and examined after 3 h (day1) and on day 7 for evaluating delayed toxicity. In separate groups Pln was administered for 5 consecutive days and examined on day 6 also after one-week delay on day12., Results: While there were only marginal differences in the locomotor tests. Immobility time during the FST significantly increased on day1 by Pln 6, 12, 40 mg/kg (185 ± 17 s, 186 ± 9 s, 172.0 ± 7 s respectively) compared with control animals (149 ± 8 s, p < 0.01), immobility time was higher than control on day 6 after multiple exposures to Pln (0.6, 6, 12, 20 mg/kg 190 ± 20s, 210 ± 4 s, 196 ± 10s, 204 ± 9 respectively, vs control 153 ± 7 p < 0.001). The immobility time remained high following a week of relapse. The co-administration of Pln with scopolamine (Scp) a MAChR antagonist reduced immobility time (141 ± 10s vs Pln 186 ± 9 s, p < 0.01)., Conclusion: Single exposure to Pln induced depressive-like effects that were reversed by Scp, indicating that MAChR stimulation may be involved. While cumulative exposures caused more pronounced changes in depressive behavior that remained after a week from the last exposure., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cholinergic neuromodulation is known to play a key role in visual working memory (VWM) - keeping relevant stimulus representations available for cognitive processes for short time periods (up to a few minutes). Despite the growing body of evidence on how the neural and cognitive mechanisms of VWM dynamically change over retention time, there is mixed evidence available on cholinergic effects as a function of VWM delay period in non-human primates. Using the delayed matching to sample VWM task in rhesus macaques (N = 6), we aimed to characterize VWM maintenance in terms of performance changes as a function of delay duration (across a wide range of delays from 1 to 76 s). Then, we studied how cholinergic neuromodulation influences VWM maintenance using the muscarinic receptor antagonist scopolamine administered alone as transient amnestic treatment, and in combination with two doses of the acetylcholinesterase inhibitor donepezil, a widely used Alzheimer's medication probing for the reversal of scopolamine-induced impairments. Results indicate that scopolamine-induced impairments of VWM maintenance are delay-dependent and specifically affect the 15-33 s time range, suggesting that scopolamine worsens the normal decay of VWM with the passage of time. Donepezil partially rescued the observed scopolamine-induced impairments of VWM performance. These results provide strong behavioral evidence for the role of increased cholinergic tone and muscarinic neuromodulation in the maintenance of VWM beyond a few seconds, in line with our current knowledge on the role of muscarinic acetylcholine receptors in sustained neural activity during VWM delay periods., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Slow transit constipation (STC) is a gastrointestinal disorder characterized by abnormal prolonged colonic transit time, which affects the life quality of many people. The decrease number of interstitial cells of Cajal (ICCs) is involved in the pathogenesis of STC. However, the molecular mechanism of loss of ICCs in STC remains unclear, making it difficult to develop new agents for the disease. In this study, we investigated the mechanism of decreasing ICCs in the pathogenesis of STC. We constructed the STC model rats by using atropine and diphenoxylate. A series of methods were used including immunofluorescence and immunochemistry staining, western blot, qRT-PCR, exosomes extraction and exosomes labeling. The results indicate that ICCs decreased in the STC rats accompanied with the macrophages activation. Further studies suggested that macrophages decreased the cell viability of ICCs by secretion exosomes containing miR-34c-5p. miR-34c5p targeted the 3Ꞌ -UTR of stem cell factor(SCF) mRNA and regulated the expression of SCF negatively. In conclusion, we demonstrated a novel regulatory mechanism of ICCs cell viability in STC. We found that exosome miR-34c-5p mediate macrophage-ICCs cross-talk. M1 macrophages derived exosomes miR-34c-5p decreased ICCs cell viability by directly targeting SCF.

Background: The dried root and rhizome of Aster tataricus (RA), is a traditional Chinese medicine has been used for more than 2000 years with the function of antitussive, expectorant and antiasthmatic. Ancient books and modern pharmacological researches demonstrated that RA may have the function of moistening intestines and relieving constipation, but there was a lack of systematic evidence. The aim of this study was to comprehensively evaluate the efficacy and possible mechanisms of ethanol extract of Aster tataricus (ATE) in treating constipation from in vivo to in vitro., Methods: In vivo, the ATE was studied in loperamide-induced constipation of mice. In vitro, different concentrations of ATE was tested separately or cumulatively on spontaneous and agonists-induced contractions of isolated rat duodenum strips., Results: In vivo, at doses of 0.16, 0.8 g/mL, ATE showed significantly promotion of the small intestinal charcoal transit, decrease of the amount of remnant fecal, and increase of the content of fecal water in colon. In addition, ATE could effectively relieve colonic pathological damage caused by loperamide as well. In vitro, with the cumulative concentration increase of ATE from 0.8 to 6.4 mg/mL, it could significantly decrease the contraction caused by KCl or Ach, and gradually restore to near base tension value.Meanwhile, it could also partially but significantly inhibit the contractions induced by Ach and CaCl 2 on rat duodenum in a concentration related manner., Conclusions: Taking all these findings together, it could be speculated that ATE may attenuate constipation mainly through antagonizing the binding of acetylcholine to muscarinic receptor, inhibiting Ca 2+ influx and anti-inflammation., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

The sense of taste provides information regarding the nutrient content, safety or potential toxicity of an edible. This is accomplished via a combination of innate and learned taste preferences. In conditioned taste aversion (CTA), rats learn to avoid ingesting a taste that has previously been paired with gastric malaise. Recent evidence points to a role of cholinergic muscarinic signaling in the amygdala for the learning and storage of emotional memories. The present study tested the participation of muscarinic receptors in the amygdala during the formation of CTA by infusing the non-specific antagonist scopolamine into the basolateral or central subnuclei before or after conditioning, as well as before retrieval. Our data show that regardless of the site of infusion, pre-conditioning administration of scopolamine impaired CTA acquisition whereas post-conditioning infusion did not affect its storage. Also, infusions into the basolateral but not in the central amygdala before retrieval test partially reduced the expression of CTA. Our results indicate that muscarinic receptors activity is required for acquisition but not consolidation of CTA. In addition, our data add to recent evidence pointing to a role of cholinergic signaling in peri-hippocampal structures in the process of memory retrieval., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microfluidic strategies combined with transduction and electronic integration have the promise of enabling miniaturized, combinatorial assays at higher speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively evaluate the transient calcium response of the endogenous Muscarinic type 1 receptor (M1) in HEK 293 T cells. The microfluidic fabrication protocol is described as well as a methodology to monitor the cell response in real time, after stimulation with M1 agonists (e.g., carbachol) and antagonists (e.g., Pirenzepine).

Over the years, experimental and clinical evidence has given support to the idea that acetylcholine (Ach) plays an essential role in mnemonic phenomena. On the other hand, the Hippocampus is already known to have a key role in learning and memory. What is yet unclear is how the Ach receptors may contribute to this brain region role during memory retrieval. The Ach receptors are divided into two broad subtypes: the ionotropic nicotinic acetylcholine receptors and the metabotropic muscarinic acetylcholine receptors. Back in 2010, we demonstrated for the first time the critical role of hippocampal α7 nicotinic acetylcholine receptors in memory reconsolidation process of an inhibitory avoidance response in mice. In the present work, we further investigate the possible implication of hippocampal muscarinic Ach receptors (mAchRs) in this process using a pharmacological approach. By specifically administrating agonists and antagonists of the different mAchRs subtypes in the hippocampus, we found that M1 and M2 but not M3 subtype may be involved in memory reconsolidation processes in mice., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Muscarinic acetylcholine receptors (mAChRs) comprise five distinct subtypes denoted M 1 to M 5 . The antagonism of M 2 subtype could increase the release of acetylcholine from vesicles into the synaptic cleft and improve postsynaptic functions in the hippocampus via M 1 receptor activation, displaying therapeutic potentials for Alzheimer's disease. However, drug development for M 2 antagonists is still challenged among different receptor subtypes. In this study, by optimizing a scaffold from virtual screening, we synthesized two focused libraries and generated up to 50 derivatives. By measuring potency and binding selectivity, we discovered a novel M 2 antagonist, ligand 47, featuring submicromolar IC 50 , high M 2 /M 4 selectivity (~30-fold) and suitable lipophilicity (cLogP = 4.55). Further study with these compounds also illustrates the structure-activity relationship of this novel scaffold. Our study could not only provide novel lead structure, which was easy to synthesize, but also offer valuable information for further development of selective M 2 ligands., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

While human eccrine sweat glands respond to adrenergic agonists, there remains a paucity of information on the factors modulating this response. Thus, we assessed the relative contribution of α- and β-adrenergic sweating during a heat exposure and as a function of individual factors of sex and training status. α- and β-adrenergic sweating was assessed in forty-eight healthy young men (n = 35) and women (n = 13) including endurance-trained (n = 12) and untrained men (n = 12) under non-heat exposure (temperate, 25°C; n = 17) and heat exposure (hot, 35°C; n = 48) conditions using transdermal iontophoresis of phenylephrine (α-adrenergic agonist) and salbutamol (β-adrenergic agonist) on the ventral forearm, respectively. Adrenergic sweating was also measured after iontophoretic administration of atropine (muscarinic receptor antagonist) or saline (control) to evaluate how changes in muscarinic receptor activity modulate the adrenergic response to a heat exposure (n = 12). α- and β-adrenergic sweating was augmented in hot compared with temperate conditions (both P ≤ .014), albeit the relative increase was greater in β (~5.4-fold)- as compared to α (~1.5-fold)-adrenergic-mediated sweating response. However, both α- and β-adrenergic sweating was abolished by atropinization (P = .001). Endurance-trained men showed an augmentation in α- (P = .043) but not β (P = .960)-adrenergic sweating as compared to untrained men. Finally, a greater α- and β-adrenergic sweating response (both P ≤ .001) was measured in habitually active men than in women. We show that heat exposure augments α-and β-adrenergic sweating differently via mechanisms associated with altered muscarinic receptor activity. Sex and training status modulate this response., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Thalamic subregions mediate various cognitive functions, including attention, inhibitory response control and decision making. Such neuronal activity is modulated by cholinergic thalamic afferents and deterioration of such modulatory signaling has been theorised to contribute to cognitive decline in neurodegenerative disorders. However, the thalamic subnuclei and cholinergic receptors involved in cognitive functioning remain largely unknown., Aims: We investigated whether muscarinic or nicotinic receptors in the mediodorsal thalamus and anterior thalamus contribute to rats' performance in the five-choice serial reaction time task, which measures sustained visual attention and impulsive action., Methods: Male Long-Evans rats were trained in the five-choice serial reaction time task then surgically implanted with guide cannulae targeting either the mediodorsal thalamus or anterior thalamus. Reversible inactivation of either the mediodorsal thalamus or anterior thalamus were achieved with infusions of the γ-aminobutyric acid-ergic agonists muscimol and baclofen prior to behavioural assessment. To investigate cholinergic mechanisms, we also assessed the behavioural effects of locally administered nicotinic (mecamylamine) and muscarinic (scopolamine) receptor antagonists., Results: Reversible inactivation of the mediodorsal thalamus severely impaired discriminative accuracy and response speed and increased omissions. Inactivation of the anterior thalamus produced less profound effects, with impaired accuracy at the highest dose. In contrast, blocking cholinergic transmission in these regions did not significantly affect five-choice serial reaction time task performance., Conclusions/interpretations: These findings show the mediodorsal thalamus plays a key role in visuospatial attentional performance that is independent of local cholinergic neurotransmission.

Objective: Crocin as an important constituent of saffron has antineuropathic pain properties; however, the exact mechanism of this effect is not known. The aim of this study was whether the hypoalgesic effect of crocin can be exerted through muscarinic receptors., Materials and Methods: In the present project, 36 male Wistar rats (200 ± 20 g) were used. Animals randomly divided into six groups (sham, neuropathy, neuropathy + crocin, neuropathy + atropine 0.5 mg/kg, neuropathy + atropine 1 mg/kg, and neuropathy + atropine 1 mg/kg + crocin). Neuropathy was induced by the chronic constriction injury (CCI) method on the sciatic nerve. Crocin and atropine was administered intraperitoneally during 14 days following the 14 th day after surgery. Pain response was detected every three days, two hours after each injection and 3 days following last injection. Mechanical allodynia and thermal hyperalgesia were detected using the Von Frey filaments and plantar test device, respectively., Results: CCI significantly reduced the paw withdrawal response to mechanical and thermal stimulus ( P < 0.01 and P < 0.05, respectively). Crocin therapy significantly reduced mechanical allodynia and thermal hyperalgesia induced by CCI ( P < 0.05). Atropine pretreatment significantly blocked the hypoalgesic effect of crocin ( P < 0.05 in mechanical allodynia and P < 0.01 in thermal hyperalgesia). Fourteen days administration of atropine alone at a dose of 0.5 mg/kg but not 1 mg/kg significantly reduced CCI-induced mechanical allodynia at day 30 after surgery., Conclusion: Crocin significantly decreased CCI-induced neuropathic pain. The hypoalgesic effect of crocin was blocked by atropine pretreatment, which indicates an important role for muscarinic receptors in the effect of crocin., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2020 Hossein Ali Safakhah et al.)

Extracellular matrix (ECM) accumulation plays a key role in the progression of bladder outlet obstruction (BOO). Muscarinic receptors have been widely reported to serve as pivotal regulators in lung tissue remodeling. However, the influence of them on human bladder smooth muscle cells (HBSMCs) and the underlying molecular mechanisms have not yet been evaluated. The purposes of the present study are to investigate the effect of muscarinic receptors on the synthesis of ECM in HBSMCs and the involvement of intracellular signal transducers. The results indicated that M 1 -M 5 muscarinic receptors were all encoded in HBSMCs. The expression rank order was M 2  > M 1  > M 5  > M 3  > M 4 . The gene and protein expression of collagen I (COL1), TIMP-1, and TIMP-2 was carbachol (CCH) concentration-dependently enhanced. The synthesis of COL1 in the supernatant of cell culture medium was significantly elevated by exposure to CCH. The CCH-induced protein expression of COL1, TIMP-1, and TIMP-2, however, was obviously reduced by the pretreatment of muscarinic receptor antagonists, atropine, and M 3 -preferring antagonist (1,1-dimethyl-4-diphenyl-acetoxypiperidinium iodide [4-DAMP]). Furthermore, ERK1/2 was activated by 100 µM CCH when compared with the control group and the pretreatment of ERK1/2 inhibitor significantly suppressed the synthesis of COL1 induced by 100 µM CCH. Besides, CCH-induced phosphorylation of ERK1/2 was remarkably restrained by the pretreatment of 4-DAMP. All in all, these findings demonstrated that M 3 receptor can modulate extracellular matrix synthesis via the ERK1/2 signaling pathway, which may provide potential novel therapeutic targets for BOO., (© 2020 Wiley Periodicals, Inc.)

Background: Urocortin 1 (Ucn1), a stress-related peptide, is a member of the corticotropin-releasing factor (CRF) family and acts as a CRF1 receptor agonist. Ucn1 and CRF1 receptor immunoreactivity are present in the enteric nervous system (ENS), and Ucn1 elicits contraction of colonic muscle strips. Considering these findings, we have hypothesized that Ucn1 acts as an excitatory neurotransmitter in the ENS. The present study was conducted to determine whether exogenously applied Ucn1 causes contractions, whether it participates in neurally mediated contraction, and whether it is released from the ENS of the rat colon., Methods: Isometric tension of the rat colonic muscle strips (middle to distal colon) in a longitudinal direction was measured. The effects of Ucn1 on phasic contractions were examined in the absence and presence of antalarmin (CRF1 receptor antagonist), tetrodotoxin (TTX), and atropine. The effects of antalarmin on electrical field stimulation (EFS)-induced contractions were examined in the absence and presence of atropine. Ucn1 peptide in the bath solution was measured after EFS using an EIA kit., Key Results: Ucn1 caused a significant and dose-dependent increase in phasic contractions. These effects were completely inhibited by antalarmin, TTX, and atropine. EFS-induced contractions were inhibited by antalarmin. Atropine markedly reduced EFS-induced contractions, and antalarmin did not decrease these contractions further. EFS elicited a significant increase in the concentration of Ucn1 in the bath solution, and this increase was completely inhibited by TTX., Conclusions and Inferences: These results suggest that Ucn1 acts as an excitatory neurotransmitter in the ENS enhancing the cholinergic neurotransmission., (© 2020 John Wiley & Sons Ltd.)

Asthma is an airway chronic inflammatory disease with significant morbidity, mortality and huge social economic burden. Previous research demonstrated that the root of Aster tataricus (RA) may have the potential to treat asthma, but the efficacy and mechanism were not clear. In this study, preliminary results in vitro showed that Fr-75 eluted from RA extract could not only completely inhibit the tracheal ring contraction raised by KCl in 20 min, but also effectively affect the tracheal ring contraction induced by KCl-, Ach- and His in a concentration-dependent manner (3.91-250 μg/mL). Further results on cells exhibited that Fr-75 could decrease the concentration of intracellular Ca 2+ as well. These results revealed the underlying mechanism in vitro that the inhibition of tracheal ring contraction might be due to the decline of the intracellular Ca 2+ concentration, which caused by suppressing calcium channel, antagonizing the muscarinic and histamine receptors. Also, results in vivo exhibited that Fr-75 could distinctly ease the symptoms of ovalbumin-sensitized mice, including relieving the pathological injury, increasing the latency to preconvulsive dyspnea and to enhanced pause, reducing the inflammatory cells, chemokines and cytokines in BALF and lung tissue. In general, it could be speculated that RA fraction may attenuate asthma through dilating the tracheal ring contraction and alleviating the lung inflammation simultaneously., (Copyright © 2020. Published by Elsevier Masson SAS.)

Novel object and social interaction tasks allow assessments of rodent cognition and social behavior. Here, we combined these tasks and defined unequivocal locations of interest. Our procedure, termed OF-NO-SI, comprised habituation to the open field (OF), novel object (NO) and social interaction (SI) stages. Habituation was measured within- and between-trials (10 minutes each, two per stage). Ambulation emerged as the appropriate proxy during the OF stage, but NO and SI trials were best quantified via direct exploration measures. We pharmacologically validated the paradigm using 5-month old C57BL/6J male mice, treated intraperitoneally with (1) 0.5 mg/kg scopolamine, (2) 0.05 mg/kg MK-801 and (3) 0.05 mg/kg SCH-23390 to block muscarinic (M1), NMDA, and D1 receptors, respectively, or (4) vehicle (distilled water). Activity and gross exploratory behavior were affected by all compounds cf. vehicle: scopolamine and MK-801 cohorts were hyperactive, while SCH-23390 caused hypo-locomotion throughout. Vehicle treated mice showed reliable habituation to all stages for time in interaction zone, directed exploration and number of visits. Exploration was severely impaired by scopolamine. MK-801 mostly affected within-session exploration but also increased exploration of the conspecific compared to the object. Interestingly, even though within-trial habituation was lacking in the SCH-23390 cohort, between-trial habituation was largely intact, despite reduced locomotion. Our data suggest that the OF-NO-SI task is a convenient and robust paradigm to measure habituation to different experimental settings and stimuli. It allows the dissociation of proxies related to activity and non-associative learning/memory, as revealed by distinct pharmacological treatment effects within- vs. between-trials.

Acetylcholine muscarinic receptors (mAChRs) contribute to both the facilitation and inhibition of levodopa-induced dyskinesia operated by striatal cholinergic interneurons, although the receptor subtypes involved remain elusive. Cholinergic afferents from the midbrain also innervate the substantia nigra reticulata, although the role of nigral mAChRs in levodopa-induced dyskinesia is unknown. Here, we investigate whether striatal and nigral M1 and/or M4 mAChRs modulate dyskinesia and the underlying striato-nigral GABAergic pathway activation in 6-hydroxydopamine hemilesioned rats. Reverse microdialysis allowed to deliver the mAChR antagonists telenzepine (M1 subtype preferring), PD-102807 and tropicamide (M4 subtype preferring), as well as the selective M4 mAChR positive allosteric modulator VU0152100 in striatum or substantia nigra, while levodopa was administered systemically. Dyskinetic movements were monitored along with nigral GABA (and glutamate) and striatal glutamate dialysate levels, taken as neurochemical correlates of striato-nigral pathway and cortico-basal ganglia-thalamo-cortical loop activation. We observed that intrastriatal telenzepine, PD-102807 and tropicamide alleviated dyskinesia and inhibited nigral GABA and striatal glutamate release. This was partially replicated by intrastriatal VU0152100. The M2 subtype preferring antagonist AFDX-116, used to elevate striatal acetylcholine levels, blocked the behavioral and neurochemical effects of PD-102807. Intranigral VU0152100 prevented levodopa-induced dyskinesia and its neurochemical correlates whereas PD-102807 was ineffective. These results suggest that striatal, likely postsynaptic, M1 mAChRs facilitate dyskinesia and striato-nigral pathway activation in vivo. Conversely, striatal M4 mAChRs can both facilitate and inhibit dyskinesia, possibly depending on their localization. Potentiation of striatal and nigral M4 mAChR transmission leads to powerful multilevel inhibition of striato-nigral pathway and attenuation of dyskinesia., Competing Interests: Declaration of Competing Interest AB is postdoc at the University of Ferrara, CAP is PhD student at the University of Ferrara, MM is employed by the University of Ferrara. The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM 1-5 ) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM 1 , hM 2 and hM 3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM 2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Opioids strongly inhibit GABAergic neurons in the rostromedial tegmental nucleus (RMTg) that expresses μ-opioid receptors to induce rewarding and psychomotor effects. M 3 and M 4 muscarinic receptors are co-localized with μ-opioid receptors at these GABAergic neurons. This study explored whether RMTg M 3 and M 4 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 M 3 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 M 4 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 M 3 muscarinic receptors are involved in opioid-induced rewarding and psychomotor effects. Therefore, RMTg M 3 muscarinic receptors may represent a promising target for the treatment of opioid addiction., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ranunculus scleratus Linn. is used in folk medicine to treat hypertension. This study was aimed at providing validation to its traditional use and to explore underlying mechanisms of action. Effects of hydro-ethanolic crude extract of the plant and its fractions on blood pressure was evaluated using direct surgical method in normotensive and in fructose induced hypertensive rats. Various doses of crude extract, RSC, (5, 10, 20, 30mg/kg) and all fractions (3, 5, 10, 20mg/kg) were studied. Results suggested that aqueous fraction of R. scleratus (RSA) produced most pronounced effects at 10mg/kg in normotensive and at 20mg/kg in hypertensive animals. Underlying mechanisms, using various pharmacological antagonists were also elucidated. Results suggested the involvement of muscarinic receptor, angiotensin converting enzyme (ACE) inhibition, ganglionic block and nitric oxide (NO) release in presenting hypotensive response.

The age and strength of fear memory are two potential parameters that can be influenced by the impairing effects of pharmacological agents on reconsolidation of fear memory. In reconsolidation, stored information is rendered labile again after being reactivated. Pharmacological manipulations at this stage result in an inability to retrieve the fear memories, suggesting that they are erased or persistently inhibited. This fear memory impairment phenomenon can be valuable to treat post-traumatic stress disorders (PTSD). Previously β-adrenergic antagonist propranolol has been repeatedly reported to impair fear memory in the treatment of PTSD. Atropine has also shown to disrupt memory formation. The present study was therefore designed to compare the effects of atropine and propranolol on reconsolidation of older fear memory in rat model of PTSD using Pavlovian fear conditioning apparatus. For this purpose 18 rats were taken and divided into control, atropine and propranolol groups and subjected to Pavlovian fear conditioning trials in order to develop animal model of PTSD. To evaluate the reconsolidation impairment of fear memory by atropine and propranolol, short term and long term memory was tested after reactivation of fear memory in rats. The present findings demonstrate that atropine significantly decreases fear expression. These results suggest that atropine significantly reduces the strength of fear memories and may be effective in the treatment of psychiatric disorders especially in PTSD., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mechanical stretch of the urothelium induces the release of ATP that activates bladder afferent nerves. In the rat urinary bladder, ATP is also a contractile co-transmitter in the parasympathetic innervation. In isolated preparations, ATP evokes a urothelial release of acetylcholine that substantially contributes to ATP-evoked contractile responses. Currently we aimed to further examine the interactions of ATP and acetylcholine in the rat urinary bladder in two in vivo models. In the whole bladder preparation, atropine reduced ATP-evoked responses by about 50% in intact but denervated bladders, while atropine had no effect after denudation of the urothelium. In a split bladder preparation, reflex-evoked responses of the contralateral half were studied by applying stimuli (agonists or stretch) to the ipsilateral half. Topical administration of ATP and methacholine as well as of stretch induced contralateral reflex-evoked contractions. While topical administration of atropine ipsilaterally reduced the ATP- and stretch-induced contralateral contractions by 27 and 39%, respectively, the P2X purinoceptor antagonist PPADS reduced them by 74 and 84%. In contrary, the muscarinic M2-(M4)-selective receptor antagonist methoctramine increased the responses by 38% (ATP) and 75% (stretch). Pirenzepine (M1-selective antagonist) had no effect on the reflex. In vitro, in the absence of the reflex, methoctramine did not affect the ATP-induced responses. It is concluded that urothelial ATP potently induces the micturition reflex and stimulates urothelial release of acetylcholine. Acetylcholine subsequently acts on afferents and on the detrusor muscle. While muscarinic M2 and/or M4 receptors in the sensory innervation exert inhibitory modulation, muscarinic M3 receptors cause excitation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study was conducted to evaluate the possible mechanisms of the relaxant effects of hydroalcoholic extract of Plantago major (P. major) on tracheal smooth muscle (TSM) in rats. The effects of cumulative concentrations of P. major (5, 10, 20 and 40 mg/mL) and theophylline (0.2, 0.4, 0.6 and 0.8 mM) were evaluated on pre-contracted TSM with 10 μΜ methacholine or 60 mM KCl. To determine the possible mechanisms, the relaxant effect of the plant was also examined on incubated TSM with atropine, indomethacin, chlorpheniramine, glibenclamide, diltiazem, papaverine, and propranolol. The results indicated concentration-dependent relaxant effects for P. major in non-incubated TSM contracted by methacholine or KCl. There was no statistically significant difference in the relaxant effects of P. major between non-incubated and incubated tissues with indomethacin, papaverine, and propranolol. However, the relaxant effects of P. major in incubated tissues with atropine (p<0.01 to p<0.001), chlorpheniramine (p<0.05 to p<0.001), glibenclamide (p<0.05), or diltiazem (p<0.01) were significantly lower than non-incubated TSM. P. major indicated relatively potent relaxant effects which were lower than those of theophylline. Muscarinic and histamine (H1) receptors inhibition, as well as calcium channel blocking and potassium channel opening effects are suggested to contribute to the TSM relaxant effect of the plant.

Cognitive impairments can be a significant problem after a traumatic brain injury (TBI), which affects millions worldwide each year. There is a need for establish reproducible cognitive assays in rodents to better understand disease mechanisms and to develop therapeutic interventions towards treating TBI-induced impairments. Our goal was to validate and standardize the radial arm water maze (RAWM) test as an assay to screen for cognitive impairments caused by TBI. RAWM is a visuo-spatial learning test, originally designed for use with rats, and later adapted for mice. The present study investigates whether test procedures, such us the presence of extra-maze cues influences learning and memory performance. C57BL/6 mice were tested in an 8-arm RAWM using a four-day protocol. We demonstrated that two days of training, exposing the mice to extra-maze cues and a visible platform, influenced learning and memory performance. Mice that did not receive training performed poorer compared to mice trained. To further validate our RAWM protocol, we used scopolamine. We, also, demonstrated that a single mild closed head injury (CHI) caused deficits in this task at two weeks post-CHI. Our data supported the use of 7 trials per day and a spaced training protocol as key factor to unmask memory impairment following CHI. Here, we provide a detailed standard operating procedure for RAWM test, which can be applied to a variety of mouse models including neurodegenerative diseases and pathology, as well as when pharmacological approaches are used., Competing Interests: The authors have declared that no competing interests exist.

We report a genomics-guided exploration of the metabolic potential of the brasilicardin producer strain Nocardia terpenica IFM 0406. Bioinformatics analysis of the whole genome sequence revealed the presence of a biosynthetic gene cluster presumably responsible for the generation of formerly unknown nocobactin derivatives. Mass spectrometry-assisted isolation led to the identification of three new siderophores, terpenibactins A (1), B (2) and C (3), which belong to the class of nocobactins. Their structures were elucidated by employing spectroscopic techniques. Compounds 1-3 demonstrated inhibitory activity towards the muscarinic M3 receptor, while exhibiting only a low cytotoxicity., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Objectives The present study was designed to investigate the effectiveness of trihexyphenidyl, a central anticholinergic drug, in preventing the post-traumatic stress disorder (PTSD) symptoms in a mouse model. Methods Mice were subjected to underwater trauma stress for 30 s on day 1 followed by three situational reminders (3rd, 7th and 14th day). Thereafter, the behavioral alterations including freezing behavior were noted on 21st day. The serum corticosterone levels were measured as a biochemical marker of trauma. Elevated plus maze test was done on day 1 and day 2 to assess the memory formation following exposure to trauma. Results Trauma and situational reminders were associated with a significant development of behavioral changes and freezing behavior on the 21st day. Moreover, there was also a significant decrease in the serum corticosterone levels. A single administration of trihexyphenidyl (2 and 5 mg/kg) significantly restored trauma associated-behavioral changes and serum corticosterone levels. Moreover, it significantly increased the transfer latency time on day 2 following stress exposure in comparison to normal mice suggesting the inhibition of memory formation during trauma exposure. Trihexyphenidyl also led to significant reduction in freezing behavior in response to situational reminders again suggesting the inhibition of formation of aversive fear memory. Conclusion The blockade of central muscarinic receptors may block the formation of aversive memory during the traumatic event, which may be manifested in form of decreased contextual fear response during situational reminders. Central anticholinergic agents may be potentially useful as prophylactic agents in preventing the development of PTSD symptoms.

Both antimuscarinics and beta-3 adrenoceptor agonists are generally used as first-line pharmacotherapy for overactive bladder (OAB). This study aimed to investigate the differences in clinical characteristics and manifestations between different medication groups using real-world data. In this retrospective study, we recruited all patients aged > 18 years diagnosed with OAB at our institute from March 2010 to December 2017. They were allocated into three groups, the antimuscarinics (group A), beta-3 adrenoceptor agonist (group B), and discontinued (group C) treatment groups, and they completed OAB symptom score and quality of life questionnaires before and after treatment. In addition, the Clinical Global Impression was recorded for treatment outcomes. A premedication urodynamic study was also applied. A total of 215 patients were analyzed (group A: 43, B: 35, C: 137). Group B was significantly older (mean age 77.4 years) than group A (69.2 years, p = 0.012) and group C (68.6 years, p = 0.001). However, there were no significant differences in sex or underlying diseases among the groups. Before treatment, there were no significant differences in the questionnaire results among all groups. The cystometric capacity of group A (mean ± SD, 257.3 ± 135.1 cm 3 ) was significantly larger than that of group B (125.8 ± 46.0 cm 3 , p = 0.002) and group C (170.5 ± 99.2 cm 3 , p = 0.001). After treatment, there were no significant differences between group A and group B in any of the questionnaire scores; however, their scores were better than those of group C. The OAB patients who adhered to antimuscarinics tended to be younger and have larger cystometric bladder capacity in the urodynamic study. However, there were no significant differences in effectiveness between the patients who took antimuscarinics and those who took a beta-3 adrenoceptor agonist.

Urgency is regarded as a core symptom of overactive bladder (OAB) and may correspond to detrusor overactivity (DO). One of the causes of OAB in men is bladder outlet obstruction (BOO) associated with benign prostatic hyperplasia (BPH). Vibegron is a novel selective β 3 -adrenoceptor agonist recently approved for the treatment of OAB. However, in OAB patients with BPH (BPH/OAB), the effects of vibegron on storage functions, especially DO and voiding functions have not been fully investigated. In this study, we evaluated the effects of a single administration of vibegron on storage function (particularly focusing on non-voiding contractions [NVC] considered a surrogate marker for DO) and voiding functions, using a rat model of partial BOO as a model for BPH/OAB. Furthermore, the utility of vibegron in combination with imidafenacin (an antimuscarinic) or silodosin (an α 1A -adrenoceptor blocker) was evaluated. Six weeks after establishment of BOO, the frequency and amplitude of NVC, evaluated by cystometrography, increased. Vibegron inhibited the frequency of NVC without affecting voiding function (micturition pressure, residual volume, and voiding efficiency). Imidafenacin and silodosin also inhibited the frequency of NVC; however, the inhibitory effects of vibegron were stronger than those of imidafenacin or silodosin. The combination of vibegron with imidafenacin or silodosin additively inhibited the frequency of NVC without worsening the voiding function. These results suggest the possibility that vibegron is effective as a single agent for the amelioration of storage symptoms in BPH/OAB patients and is useful in combination with either antimuscarinics or α 1 -adrenoceptor blockers., Competing Interests: Declaration of competing interest The authors are employees of Kissei Pharmaceutical Co., Ltd., and Kyorin Pharmaceutical Co., Ltd. These companies funded and approved the conduct of the study. The authors conducted the study at their own discretion and have editorial freedom with respect to the manuscript. The authors will not receive any monetary reward from either company upon acceptance of the manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The effect of acetylcholine (ACh) on pacemaking and spontaneous contractions in the gastrointestinal tract is not well characterized. The current study aims to profile the effect of several muscarinic and nicotinic receptor agonists and antagonists on pacemaker potentials in the ICR mouse ileum. Pacemaker potentials of whole thickness mouse ileal segments were recorded extracellularly using a 60-channel microelectrode array (MEA) platform. A spatiotemporal analysis integrated the frequency, amplitude, and velocity measurements of pacemaker currents. Comparative data were obtained by recording spontaneous smooth muscle tone in a conventional organ bath. On the MEA, ACh (0.3-300 μM) and bethanechol (0.3-300 μM) significantly reduced ileal pacemaker potentials. The inhibitory effect of ACh was mimicked by donepezil (300 μM) but not nicotine (0.3-7 mM). Atropine (300 μM), but not hexamethonium (300 μM), reversed the inhibitory actions of ACh and bethanechol and revealed excitatory properties manifested as increases in pacemaker frequency. A spatial analysis also revealed that atropine, but not hexamethonium, reversed the ACh-induced distortion of pacemaker propagation activity. Atropine (0.001-3 mM) and hexamethonium (0.3-7 mM) alone were inactive. In the organ bath, ACh (300 nM) and bethanechol (30 μM) induced ileal tonic contractions, while inhibiting basal spontaneous contractions at 300 μM. Atropine (1 μM), but not hexamethonium (1-300 μM), reversed both the tonic contractions and the inhibition of the spontaneous contractions of ACh and bethanechol and revealed an excitatory effect manifested as an increasing in the frequency of contractions. Muscarinic, but not nicotinic, receptors appear to mediate the inhibitory actions of ACh on mouse ileal pacemaker potentials. NEW & NOTEWORTHY The study discovered an acute action of acetylcholine on pacemaker potentials that is mediated by muscarinic receptors on the mouse ileum. Bethanechol, but not nicotine, mimicked the inhibitory actions of acetylcholine on pacemaker potentials. Atropine, but not hexamethonium, reversed the inhibitory actions of acetylcholine. When introduced after acetylcholine, atropine exhibited excitatory actions that increased the pacemaker frequency. Acetylcholine and bethanechol distorted the propagation activity and pattern, and this was also reversed by atropine. These actions of acetylcholine on pacemaker potentials may contribute to pathophysiology in bowel diseases.

Background: overactive bladder (OAB) affects 17-41% older adults in community dwelled setting. For several years, antimuscarinics have been validated as the first-line medical treatment for OAB. Despite abundant data obtained from clinical trials provisions the use of antimuscarinics, investigation about the effect of this drug on cognitive function in elderly remains scarce. The objective of this study is to investigate the effect of antimuscarinics therapy on cognitive functions in OAB geriatric patients., Methods: this study design is a systematic review and meta-analysis. Studies were collected using several search engines; those were PubMed, Science Direct, Cochrane, and EBSCOhost using predetermined MeSH keywords with Boolean operators. Selection of studies was done by three reviewers. Studies which fulfilled the inclusion and exclusion criteria underwent full-text review. For every selected full text, we extracted the following data if available: patients demographics, types of antimuscarinics used, placebo, dose, follow-up period, and Mini-Mental State Examination (MMSE) total score., Results: a total of 8 studies from an initial 146 publications were selected. There were 8 antimuscarinic agents evaluated in the studies, including Oxybutynin, Darifenacin, Tolterodine, Trospium, Imidafenacin, Propiverine hydrochloride, Fesoterodine, and Solifenacin. Oxybutynin was shown to have largest effect towards the decline of MMSE score [Mean difference: -2.90; 95% CI: -4.07, -1.73]. Darifenacin and Tolterodine were also shown to be significant in the decline of total MMSE score, although still inferior to Oxybutynin., Conclusion: the use of most antimuscarinics medication has little to no effect towards the cognitive function in the management of overactive bladder in elderly patients. However, Oxybutynin, Darifenacin, and Tolterodine was shown to have significant decrease in cognitive functions, as shown in the decline of total MMSE score.

Introduction: Overactive bladder is a prevalent symptom complex that affects the patient's quality of life. Any disruption between the neuronal micturition pathway can lead to bladder overactivity. Neurogenic causes, myogenic causes, aging, bladder outlet obstruction, sex, and psychological factors are some of the factors contributing to bladder overactivity. The complaint of any symptoms of OAB, which is highly prevalent and affects overall QOL, often needs therapeutic interventions. When conservative therapy methods fail, the addition of medications is recommended. The most commonly used agents for the treatment of OAB are antimuscarinic drugs. New classes of drugs, such as beta-3 agonists, have enriched our pharmacologic armamentarium., Areas Covered: In this review, with a special focus on oral pharmacological treatments, we discussed the definition, etiology, symptoms, diagnosis, and management of OAB., Expert Opinion: OAB is a multifactorial condition with every patient presenting with a different collection of symptoms and signs. Medical therapies should be given in conjunction with behavioral therapies. Using high or low doses, flexible doses, and stopping or changing the medications are interchangeable strategies based on the level of treatment efficacy and patient satisfaction.

Acetylcholine is implicated in mood disorders including depression and anxiety. Increased cholinergic tone in humans and rodents produces pro-depressive and anxiogenic-like effects. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate these responses in male rats, as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and the forced swim test (FST). However, these effects have not been examined in females, and the VTA muscarinic receptor subtype(s) mediating the pro-depressive and anxiogenic-like behavioral effects of increased cholinergic tone are unknown. We first examined the behavioral effects of increased VTA cholinergic tone in male and female rats, and then determined whether VTA muscarinic M5 receptors were mediating these effects. VTA infusion of the acetylcholinesterase inhibitor physostigmine (0.5 μg, 1 μg and 2 μg/side) in males and females produced anhedonic-like, anxiogenic, pro-depressive-like responses on the SPT, EPM, and FST. In females, VTA administration of the muscarinic M5 selective negative allosteric modulator VU6000181 (0.68 ng, 2.3 ng, 6.8 ng/side for a 3 μM, 10 μM, 30 μM/side infusion) did not alter SPT, EPM nor FST behavior. However, in males intra-VTA infusion of VU6000181 alone reduced time spent immobile on the FST. Furthermore, co-infusion of VU6000181 with physostigmine, in male and female rats, attenuated the pro-depressive and anxiogenic-like behavioral responses induced by VTA physostigmine alone, in the SPT, EPM, and FST. Together, these data reveal a critical role of VTA M5 receptors in mediating the anhedonic, anxiogenic, and depressive-like behavioral effects of increased cholinergic tone in the VTA., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Muscarinic antagonists promote sensory neurite outgrowth in vitro and prevent and/or reverse multiple indices of peripheral neuropathy in rodent models of diabetes, chemotherapy-induced peripheral neuropathy, and HIV protein-induced neuropathy when delivered systemically. We measured plasma concentrations of the M 1 receptor-selective muscarinic antagonist pirenzepine when delivered by subcutaneous injection, oral gavage, or topical application to the skin and investigated efficacy of topically delivered pirenzepine against indices of peripheral neuropathy in diabetic mice. Topical application of 2% pirenzepine to the paw resulted in plasma concentrations 6 hours postdelivery that approximated those previously shown to promote neurite outgrowth in vitro. Topical delivery of pirenzepine to the paw of mice with streptozotocin-induced diabetes dose-dependently (0.1%-10.0%) prevented tactile allodynia, thermal hypoalgesia, and loss of epidermal nerve fibers in the treated paw and attenuated large fiber motor nerve conduction slowing in the ipsilateral limb. Efficacy against some indices of neuropathy was also noted in the contralateral limb, indicating systemic effects following local treatment. Topical pirenzepine also reversed established paw heat hypoalgesia, whereas withdrawal of treatment resulted in a gradual decline in efficacy over 2-4 weeks. Efficacy of topical pirenzepine was muted when treatment was reduced from 5 to 3 or 1 day/wk. Similar local effects were noted with the nonselective muscarinic receptor antagonist atropine when applied either to the paw or to the eye. Topical delivery of muscarinic antagonists may serve as a practical therapeutic approach to treating diabetic and other peripheral neuropathies. SIGNIFICANCE STATEMENT: Muscarinic antagonist pirenzepine alleviates diabetic peripheral neuropathy when applied topically in mice., Competing Interests: P.F. and L.P.K. are cofounders, shareholders, and serve on the Board of Directors of Winsantor Inc. N.A.C. is a cofounder and shareholder in Winsantor Inc. K.E.F. is an employee of Winsantor Inc. and UCSD., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Our previous study showed that kaempferitrin, the main flavonoid from Bauhinia forficata Link leaves, induces diuresis and saluresis when orally given to rats. Since afzelin (AFZ) and kaempferol (KFL) are active compounds from the biometabolism of kaempferitrin, the diuretic and renal protective properties of these two compounds were evaluated. While the acute treatment with AFZ evoked a diuretic action associated with an increase in Cl - excretion and a Ca 2+ -sparing effect, KFL did not present any activity. The pretreatment with a muscarinic receptor blocker or with an inhibitor of the cyclooxygenase fully avoided AFZ-induced diuresis. AFZ also induced a prolonged (7-day treatment) diuretic effect in normotensive (NTR) and hypertensive rats (SHR), with an increase of urinary Na + and Cl - excretion, while it decreased the elimination of Ca 2+ . AFZ was able to decrease ROS and nitrite generation on kidney homogenates in comparison with the SHR group treated with the vehicle, as well as mitigated the changes in the renal corpuscle region (glomerulus and Bowman's capsule). Moreover, AFZ significantly reduced calcium oxalate crystal formation in urine, with inhibition rates of 41% for the NTR and 92% for the SHR group. Taken together, this study shows that AFZ exerts acute and prolonged diuretic effects plus protective renal properties.

A series of novel 1,4-dioxane analogues of the muscarinic acetylcholine receptor (mAChR) antagonist 2 was synthesized and studied for their affinity at M 1 -M 5 mAChRs. The 6-cyclohexyl-6-phenyl derivative 3b , with a cis configuration between the CH 2 N + (CH 3 ) 3 chain in the 2-position and the cyclohexyl moiety in the 6-position, showed p K i 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 (2 S ,6 S )-(-)- 3b and (2 S ,6 S )-(-)- 33b , respectively. Docking simulations on the M 3 mAChR-resolved structure rationalized the experimental observations. The quaternary ammonium function, which should prevent the crossing of the blood-brain barrier, and the high M 3 /M 2 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 M 3 receptors are involved.

Introduction: The increased acetylcholine signaling in asthma pathophysiology offers the rationale for the use of LAMAs in the treatment of asthmatic patients. Tiotropium is still the only LAMA approved for use in asthma but there is a real interest in developing novel LAMAs for the treatment of asthma, or at least to extend this indication to other LAMAs already on the market., Areas Covered: We examined and discussed trials and research that have studied or are evaluating the role of LAMAs already on the market in asthma and possible novel muscarinic acetylcholine receptor antagonists., Expert Opinion: Glycopyrronium and umeclidinium will soon be included in the GINA strategy with the same current indications of tiotropium. It is likely that the choice of the LAMA will be influenced not so much by its pharmacological profile as by the type of triple therapy chosen. It is extremely difficult to identify a new LAMA that is more effective than tiotropium, but is it plausible that new technologies that will allow delivering the drug in a more targeted way and with a lower risk of adverse effects may represent the real progress in the use of LAMAs in asthma in the coming years.

Despite increased awareness and intensified biomedical research efforts, the prevalence of obesity continues to rise worldwide. This is alarming, because obesity accelerates the progression of several chronic disorders, including type 2 diabetes, cancer and cardiovascular disease. Individuals who experience significant weight loss must combat powerful counter-regulatory energy homeostatic processes, and, typically, most individuals regain the lost weight. Therefore, decoding the neural mechanisms underlying the regulation of energy homeostasis is necessary for developing breakthroughs in obesity management. It has been known for decades that cholinergic neurotransmission both directly and indirectly modulates energy homeostasis and metabolic health. Despite this insight, the molecular details underlying the modulation remain ill-defined, and the potential for targeting cholinergic muscarinic receptors for treating metabolic disease is largely uncharted. In this MiniReview, we scrutinize the literature that has formed our knowledge of muscarinic acetylcholine receptors (mAChRs) in energy homeostasis. The role of mAChRs in canonical appetite-regulating circuits will be discussed as will the more indirect regulation of energy homoeostasis via neurocircuits linked to motivated behaviours and emotional states. Finally, we discuss the therapeutic prospects of targeting mAChRs for the treatment of obesity and type 2 diabetes., (© 2019 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

Racemic 3-quinuclidinyl-α-methoxydiphenylacetate (MB266) was synthesised. Its activity at muscarinic acetylcholine receptors (mAChRs), and muscle and neuronal nicotinic acetylcholine receptors (nAChRs), was compared to that of atropine and racemic 3-quinucidinyl benzilate (QNB) using a functional assay based on agonist-induced elevation of intracellular calcium ion concentration in CN21, Chinese Hamster Ovary (CHO) and SHSY5Y human cell lines. MB266 acted as an antagonist at acetylcholine receptors, displaying 18-fold selectivity for mAChR versus nAChR (compared to the 15,200-fold selectivity observed for QNB). Thus O-methylation of QNB reduced the affinity for mAChR antagonism and increased the relative potency at both muscle and neuronal nAChRs. Despite MB266 having a pharmacological profile potentially useful for the treatment of anticholinesterase poisoning, its administration did not improve the neuromuscular function in a soman-poisoned guinea-pig diaphragm preparation pretreated with the organophosphorus nerve agent soman. Consideration should be given to exploring the potential of MB266 for possible anticonvulsant action in vitro as part of a multi-targeted ligand approach., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

The purpose of this study was to explore whether individual differences in glucocorticoid concentrations were associated with symptom improvement following exposure therapy for patients with social anxiety disorder. To do this, 60 participants with social anxiety disorder completed a randomized-controlled trial of exposure therapy, where participants were randomized to receive scopolamine-augmentation or placebo during their 7 exposure sessions. Scopolamine is an antimuscarinic which blocks the effects of acetylcholine and reduces autonomic arousal. During sessions 1, 4, 7, and during the post-treatment extinction assessment, participants provided up to 16 saliva samples (4 in each session). Pre-treatment, post-treatment, and at 1-month follow-up, participants completed the Liebowitz Social Anxiety Scale to monitor change in fear and avoidance symptoms. Elevated endogenous in-session cortisol during exposure sessions was associated with less symptom improvement from pre- to post-treatment and at 1-month follow-up. The association between elevated endogenous in-session cortisol and attenuated symptom change was not moderated by scopolamine treatment condition. Individuals with social anxiety disorder who have elevated neuroendocrine signaling may under-benefit from exposure therapy. This is the first study, to our knowledge, to examine whether endogenous in-session cortisol concentrations predict symptom changes following exposure therapy for the treatment of social anxiety disorder. More investigation of non-invasive and reliable biological markers that explain variability in responses to effective treatments are needed., Competing Interests: Declaration of Competing Interest The authors report no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

This study aimed to use central and peripheral assays to compare the effects of the muscarinic antagonist scopolamine with those of a novel muscarinic antagonist, L-687,306 [(3R,4R)-3-(3-cyclopropyl-1,2,4,oxadiazol[5-yl]-1-azabicyclo[2.2.1]heptane. Groups of rats were trained to discriminate the stimulus effects of the muscarinic agonist, arecoline (1.0 mg/kg); concomitant measures of response rate were recorded. Separate groups were prepared with telemetery devices for recording bradycardia induced by arecoline (10 mg/kg). Methyl arecoline and arecoline were nearly equally potent in producing a brief but profound bradycardia, indicative of an equivalent effect in the heart. L-687,306 and scopolamine were both able to block this peripheral effect of arecoline. L-687,306 produced a surmountable antagonism of both the discriminative and rate-suppressing effects of arecoline. Scopolamine, however, was unable to antagonize the rate-reducing effects of arecoline in the discrimination assay. This limited the number of rats that could respond to the discriminative stimulus effects of arecoline, as well as the amount of arecoline stimulus effects they were able to report. The data suggest that L-687,306 may be a more generally effective muscarinic antagonist than scopolamine and support earlier reports that this antagonist has less direct effect on behavior.

Introduction: In chronic obstructive pulmonary disease (COPD), inhaled long-acting antimuscarinic agents (LAMA) are effective maintenance therapies used across all severity stages of the disease. Most of them are administered via dry powder inhalers, but these devices require a potent inspiratory flow which cannot be effectively achieved by patients with advanced disease. In such patients, inhaled therapy via nebulization might be an option., Areas Covered: Revefenacin is a LAMA that was specifically formulated for once daily nebulization and which was authorized by the FDA as a maintenance therapy for COPD. In phase II and III clinical studies discussed in this review, revefenacin demonstrated its rapid onset of action and sustained effect on lung function on both a short- and long-term basis., Expert Opinion: Nebulized revefenacin with once daily use does not require any particular effort of administration and hence can be used by patients with severe airways obstruction or by those having milder cognitive deficits. Further studies are needed, however, to better document the long-term cardiovascular safety and its ability to reduce the exacerbation rate.

Mitochondrial dysfunction is implicated in a variety of neurodegenerative diseases of the nervous system. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a regulator of mitochondrial function in multiple cell types. In sensory neurons, AMP-activated protein kinase (AMPK) augments PGC-1α activity and this pathway is depressed in diabetes leading to mitochondrial dysfunction and neurodegeneration. Antimuscarinic drugs targeting the muscarinic acetylcholine type 1 receptor (M 1 R) prevent/reverse neurodegeneration by inducing nerve regeneration in rodent models of diabetes and chemotherapy-induced peripheral neuropathy (CIPN). Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ) is an upstream regulator of AMPK activity. We hypothesized that antimuscarinic drugs modulate CaMKKβ to enhance activity of AMPK, and PGC-1α, increase mitochondrial function and thus protect from neurodegeneration. We used the specific M 1 R antagonist muscarinic toxin 7 (MT7) to manipulate muscarinic signaling in the dorsal root ganglia (DRG) neurons of normal rats or rats with streptozotocin-induced diabetes. DRG neurons treated with MT7 (100 nM) or a selective muscarinic antagonist, pirenzepine (1 μM), for 24 h showed increased neurite outgrowth that was blocked by the CaMKK inhibitor STO-609 (1 μM) or short hairpin RNA to CaMKKβ. MT7 enhanced AMPK phosphorylation which was blocked by STO-609 (1 μM). PGC-1α reporter activity was augmented up to 2-fold (p < 0.05) by MT7 and blocked by STO-609. Mitochondrial maximal respiration and spare respiratory capacity were elevated after 3 h of exposure to MT7 (p < 0.05). Diabetes and CIPN induced a significant (p < 0.05) decrease in corneal nerve density which was corrected by topical delivery of MT7. We reveal a novel M 1 R-modulated, CaMKKβ-dependent pathway in neurons that represents a therapeutic target to enhance nerve repair in two of the most common forms of peripheral neuropathy.

Introduction: Remyelination is a highly effective regenerative process that can restore axon function, prevent axonal loss, and reverse clinical deficits after demyelination. Hence, the promotion of remyelination is a logical goal in patients with multiple sclerosis (MS) in which remyelination is often insufficient. However, despite great progress regarding the development of immunomodulatory therapies for MS and an abundance of promising evidence from preclinical experiments so far, no therapy has convincingly demonstrated clinically significant remyelination properties. Therefore, enhancing myelin repair is an urgent and unmet need in MS., Areas Covered: We searched clinicaltrials.gov and pubmed.ncbi.nlm.nih.gov and focused on therapeutic agents in development from the preclinical stage to clinical phase II. We selected agents for which data are available from in vitro experiments and at least one toxic demyelination animal model that reached at least phase I in clinical development in MS patients., Expert Opinion: The evidence to promote remyelination is very promising for several agents, some of which possess anti-muscarinergic properties. Since remyelination is a complex process that involves various coordinated steps, a combination of different therapeutic approaches addressing different aspects of this regenerative mechanism may be reasonable. Furthermore, suitable surrogate markers of remyelination are necessary for proof-of-concept clinical trials.

In a similar manner to other learning paradigms, intact muscarinic acetylcholine receptor (mAChR) neurotransmission or protein synthesis regulation in the anterior insular cortex (aIC) is necessary for appetitive taste learning. Here we describe a parallel local molecular pathway, where GABA A receptor control of mAChR activation causes upregulation of miRNA-182 and quinone reductase 2 (QR2) mRNA destabilization in the rodent aIC. Damage to long-term memory by prevention of this process, with the use of mAChR antagonist scopolamine before novel taste learning, can be rescued by local QR2 inhibition, demonstrating that QR2 acts downstream of local muscarinic activation. Furthermore, we prove for the first time the presence of endogenous QR2 cofactors in the brain, establishing QR2 as a functional reductase there. In turn, we show that QR2 activity causes the generation of reactive oxygen species, leading to modulation in Kv2.1 redox state. QR2 expression reduction therefore is a previously unaccounted mode of mAChR-mediated inflammation reduction, and thus adds QR2 to the cadre of redox modulators in the brain. The concomitant reduction in QR2 activity during memory consolidation suggests a complementary mechanism to the well established molecular processes of this phase, by which the cortex gleans important information from general sensory stimuli. This places QR2 as a promising new target to tackle neurodegenerative inflammation and the associated impediment of novel memory formation in diseases such as Alzheimer's disease., (Copyright © 2020 Gould et al.)