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Typical agonists of G protein-coupled receptors (GPCRs), including muscarinic acetylcholine receptors (mAChRs), activate both G-protein and β-arrestin signaling systems, and are termed balanced agonists. In contrast, biased agonists selectively activate a single pathway, thereby offering therapeutic potential for the specific activation of that pathway. The mAChR agonists carbachol and pilocarpine are known to induce phosphorylation of extracellular signal-regulated kinase-1/2 (ERK1/2) via G-protein-dependent and -independent pathways, respectively. We investigated the involvement of β-arrestin and its downstream mechanisms in the ERK1/2 phosphorylation induced by carbachol and pilocarpine in the human salivary ductal cell line, HSY cells. HSY cells were stimulated with pilocarpine or carbachol, with or without various inhibitors. The cell lysates were analyzed by western blotting using the antibodies p44/p42MAPK and phosphor-p44/p42MAPK. Western blot analysis revealed that carbachol elicited greater stimulation of ERK1/2 phosphorylation compared to pilocarpine. ERK1/2 phosphorylation was inhibited by atropine and gefitinib, suggesting that mAChR activation induces transactivation of epidermal growth factor receptors (EGFR). Moreover, inhibition of carbachol-mediated ERK1/2 phosphorylation was achieved by GF-109203X (a PKC inhibitor), a βARK1/GRK2 inhibitor, barbadin (a β-arrestin inhibitor), pitstop 2 (a clathrin inhibitor), and dynole 34-2 (a dynamin inhibitor). In contrast, pilocarpine-mediated ERK1/2 phosphorylation was only inhibited by barbadin (a β-arrestin inhibitor) and PP2 (a Src inhibitor). Carbachol activates both G-protein and β-arrestin pathways, whereas pilocarpine exclusively activates the β-arrestin pathway. Additionally, downstream of β-arrestin, carbachol activates clathrin-dependent internalization, while pilocarpine activates Src. [Display omitted] • Muscarinic receptors agonists carbachol and pilocarpine induce phosphorylation of ERK1/2. • Carbachol activates two pathways, the G protein- and β-arrestin-mediated pathways. • Pilocarpine preferentially activates β-arrestin-mediated pathways. • Carbachol and pilocarpine activate different pathways downstream of β-arrestin in HSY cells. [ABSTRACT FROM AUTHOR]

Demyelination affects the propogation of neuronal action potential by slowing down the progression. This process results in a neuro-impairment like Multiple Sclerosis (MS). Evidence show that MS also contributes to involvement of the autonomic system. In the molecular approach to this involvement, we aimed to observe muscarinic ACh receptor 2–3 (mAChR2–3), and inwardly rectifying potassium channel 3.1 (Kir3.1) immunoreactivities on the brainstem, vagus nerve, and heart under cuprizone model. Wistar albino rats were randomly divided into 8 groups; duplicating 4 groups as male and female: control groups (n = 3 +3), Cuprizone groups (n = 12 +12), sham groups (n = 4 +4), and carboxy-methyl-cellulose groups (n = 3 +3). Cuprizone-fed rats underwent demyelination via Luxol fast blue (LFB) staining of the hippocampus (Gyrus dentatus and Cornu Ammonis) and cortex. Immunohistochemistry analysis followed to the pathologic measurement of the brainstem, vagus nerve, and heart for mAChR2, mAChR3 and Kir3.1 proteins A significant demyelination was observed in the hippocampus and cortex tissues of rats in the female and male cuprizone groups. Myelin basic protein immunoreactivity demonstrated that cuprizone groups, in both males and females, had down-regulation in the hippocampus and cortex areas. The weights of the cuprizone-fed rats significantly decreased over six weeks. Dilated blood vessels and neuronal degeneration were severe in the hippocampus and cortex of the cuprizone groups. In the female cuprizone group, expression of mAChR2 and mAChR2 was significantly increased in the brainstem, atrium/ventricle of heart, and left/right sections of vagus nerve. Kir3.1 channels were also up-regulated in the left vagus nerve and heart sections of the female cuprizone group Especially in our data where female-based significant results were obtained reveal that demyelination may lead to significant mAChR2, mAChR3 and Kir3.1 changes in brainstem, vagus nerve, and heart. A high immunoreactive response to demyelination at cholinergic centers may be a new target • Cuprizone-induced demyelination showed affecting autonomic tissues as brainstem. • Dilated blood vessels and neuron degeneration was found in the hippocampus and cortex. • Muscarinic receptor 2–3 expressions were increased in female demyelinating rats. • The heart responded better in immunohistology than the brainstem and vagus nerve. [ABSTRACT FROM AUTHOR]

There is an accumulating body of evidence implicating the muscarinic acetylcholine receptor 4 (M 4) in schizophrenia and dementia with Lewy bodies, however, a clinically validated M 4 positron emission tomography (PET) radioligand is currently lacking. As such, the aim of this study was to develop a suitable M 4 PET ligand that allows the non-invasive visualization of M 4 in the brain. Structure–activity relationship studies of pyrazol-4-yl-pyridine derivates led to the discovery of target compound 12 ― a subtype-selective positive allosteric modulator (PAM). The radiofluorinated analogue, [18F] 12 , was synthesized in 28 ± 10% radiochemical yield, >37 GBq/μmol and an excellent radiochemical purity >99%. Initial in vitro autoradiograms on rodent brain sections were performed in the absence of carbachol and showed moderate specificity as well as a low selectivity of [18F] 12 for the M 4 -rich striatum. However, in the presence of carbachol, a significant increase in tracer binding was observed in the rat striatum, which was reduced by >60% under blocking conditions, thus indicating that orthosteric ligand interaction is required for efficient binding of [18F] 12 to the allosteric site. Remarkably, however, the presence of carbachol was not required for high specific binding in the non-human primate (NHP) and human striatum, and did not further improve the specificity and selectivity of [18F] 12 in higher species. These results pointed towards significant species-differences and paved the way for a preliminary PET study in NHP, where peak brain uptake of [18F] 12 was found in the putamen and temporal cortex. In conclusion, we report on the identification and preclinical development of the first radiofluorinated M 4 PET radioligand with promising attributes. The availability of a clinically validated M 4 PET radioligand harbors potential to facilitate drug development and provide a useful diagnostic tool for non-invasive imaging. This work describes the synthesis and preclinical evaluation of a radiofluorinated molecular imaging probe for the subtype-selective visualization of muscarinic acetylcholine receptors (M 4) with positron emission tomography (PET). [Display omitted] [ABSTRACT FROM AUTHOR]

• Muscarinic acetylcholine receptors (mAChR) inhibit KCNQ channels in calyces. • Activation of mAChRs has an excitatory effect on calyx terminals. • Activation of mAChRs decreases the first spike latency during step depolarizations. • Activation of mAChRs decreases the action potential threshold. • mAChR activation by efferents can be used to modulate calyx activity. Vestibular nerve afferents are divided into regular and irregular groups based on the variability of interspike intervals in their resting discharge. Most afferents receive inputs from bouton terminals that contact type II hair cells as well as from calyx terminals that cover the basolateral walls of type I hair cells. Calyces have an abundance of different subtypes of KCNQ (Kv7) potassium channels and muscarinic acetylcholine receptors (mAChRs) and receive cholinergic efferent inputs from neurons in the brainstem. We investigated whether mAChRs affected membrane properties and firing patterns of calyx terminals through modulation of KCNQ channel activity. Patch clamp recordings were performed from calyx terminals in central regions of the cristae of the horizontal and anterior canals in 13–26 day old Sprague-Dawley rats. KCNQ mediated currents were observed as voltage sensitive currents with slow kinetics (activation and deactivation), resulting in spike frequency adaptation so that calyces at best fired a single action potential at the beginning of a depolarizing step. Activation of mAChRs by application of oxotremorine methiodide or inhibition of KCNQ channels by linopirdine dihydrochloride decreased voltage activated currents by ∼30%, decreased first spike latencies by ∼40%, resulted in action potential generation in response to smaller current injections and at lower (i.e., more hyperpolarized) membrane potentials, and increased the number of spikes fired during depolarizing steps. Interestingly, some of the calyces showed spontaneous discharge in the presence of these drugs. Together, these findings suggest that cholinergic efferents can modulate the response properties and encoding of head movements by afferents. [ABSTRACT FROM AUTHOR]

Receptor occupancy is an indicator of antipsychotic efficacy and safety. It is desirable to simultaneously determine the occupancy of multiple brain receptors as an indicator of the efficacy and central side effects of antipsychotics because many of these drugs have binding affinities for various receptors, such as dopamine 2 (D 2), histamine 1 (H 1), and muscarinic acetylcholine (mACh) receptors. The purpose of this study was to develop a method for the simultaneous measurement of multiple receptor occupancies in the brain by the simultaneous quantification of unlabeled tracer levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Rats were pre-administered with a vehicle, displacer, or olanzapine, and mixed solutions of raclopride, doxepin, and 3-quinuclidinyl benzilate (3-QNB) were administered (3, 10, and 30 μg/kg). The brain tissue and plasma tracer concentrations were quantified 45 min later using LC-MS/MS, and the binding potential was calculated. The highest binding potential was observed at 3 μg/kg raclopride, 10 μg/kg doxepin, and 30 μg/kg 3-QNB. Tracer-specific binding at these optimal tracer doses in the cerebral cortex was markedly reduced by pre-administration of displacers. D 2 , H 1, and mACh receptor occupancy by olanzapine increased in a dose-dependent manner, reaching 70–95%, 19–43%, and 12–45%, respectively, at an olanzapine dose range of 3–10 mg/kg. These results suggest that simultaneous determination of in vivo D 2 , H 1 , and mACh receptor occupancy is possible using LC-MS/MS. [ABSTRACT FROM AUTHOR]

Muscarinic acetylcholine receptors (mAChR) play important roles in regulating complex behaviors such as cognition, movement, and reward, making them ideally situated as potential drug targets for the treatment of several brain disorders. Recent advances in the discovery of subtype-selective allosteric modulators for mAChRs has provided an unprecedented opportunity for highly specific modulation of signaling by individual mAChR subtypes in the brain. Recently, mAChR allosteric modulators have entered clinical development for Alzheimer's disease (AD) and schizophrenia, and have potential utility for other brain disorders. However, mAChR allosteric modulators can display a diverse array of pharmacological properties, and a more nuanced understanding of the mAChR will be necessary to best translate preclinical findings into successful clinical treatments. mAChR allosteric modulators demonstrate unparalleled subtype selectivity, can possess a wide array of distinct pharmacological properties, and are rapidly advancing into the clinic for the treatment of multiple central nervous system disorders. M 1 mAChR positive allosteric modulators (PAMs) may enhance cognition and reverse memory deficits in AD and schizophrenia, and may display a larger therapeutic window than acetylcholinesterase inhibitors. M 4 PAMs can reduce dopamine release and demonstrate antipsychotic-like effects in preclinical animal models. Recent preclinical literature suggests that M 5 negative allosteric modulators may effectively treat an array of substance use disorders without reducing the effects of natural rewards. [ABSTRACT FROM AUTHOR]

This letter describes progress towards an M 4 PAM preclinical candidate inspired by an unexpected aldehyde oxidase (AO) metabolite of a novel, CNS penetrant thieno[2,3- c ]pyridine core to an equipotent, non-CNS penetrant thieno[2,3- c ]pyrdin-7(6 H)-one core. Medicinal chemistry design efforts yielded two novel tricyclic cores that enhanced M 4 PAM potency, regained CNS penetration, displayed favorable DMPK properties and afforded robust in vivo efficacy in reversing amphetamine-induced hyperlocomotion in rats. [ABSTRACT FROM AUTHOR]

Drug relapse after periods of abstinence is a common feature of substance abuse. Moreover, anxiety and other mood disorders are often co-morbid with substance abuse. Cholinergic receptors in the ventral tegmental area (VTA) are known to mediate drug-seeking and anxiety-related behavior in rodent models. However, it is unclear if overlapping VTA cholinergic mechanisms mediate drug relapse and anxiety-related behaviors associated with drug abstinence. We examined the effects of VTA cholinergic receptor blockade on cue-induced cocaine seeking and anxiety during cocaine abstinence. Male Sprague–Dawley rats were trained to self-administer intravenous cocaine (~ 0.5 mg/kg/infusion, FR1 schedule) for 10 days, followed by 14 days of forced abstinence. VTA infusion of the non-selective nicotinic acetylcholine receptor antagonist mecamylamine (0, 10, and 30 μg/side) or the non-selective muscarinic receptor antagonist scopolamine (0, 2.4 and 24 μg /side) significantly decreased cue-induced cocaine seeking. In cocaine naïve rats, VTA mecamylamine or scopolamine also led to dose-dependent increases in open arm time in the elevated plus maze (EPM). In contrast, rats that received I.V. cocaine, compared to received I.V. saline rats, displayed an anxiogenic response on day 14 of abstinence as reflected by decreased open arm time in the EPM. Furthermore, low doses of VTA mecamylamine (10 μg /side) or scopolamine (2.4 μg /side), that did not alter EPM behavior in cocaine naive rats, were sufficient to reverse the anxiogenic effects of cocaine abstinence. Together, these data point to an overlapping role of VTA cholinergic mechanisms to regulate relapse and mood disorder-related responses during cocaine abstinence. • VTA mAChR and nAChR blockade attenuates cue-induced cocaine seeking on forced abstinence day 14. • VTA mAChR and nAChR blockade produces an anxiolytic-like effect on the elevated plus maze in cocaine naive rats. • I.V. cocaine self-administration and forced abstinence produces an anxiogenic-like effect on the elevated plus maze. • VTA mAChR and nAChR blockade attenuates the cocaine-induced anxiogenic-like effect on the elevated plus maze. [ABSTRACT FROM AUTHOR]

• First disclosure of VU6005806/AZN-00016130, an M 4 PAM profiled as a putative candidate. • Novel SAR findings based on novel variations to the C3 position on the pyridazine core. • Minimum effective dose of 0.56 mg/kg po in rat AHL. This letter describes progress towards an M 4 PAM preclinical candidate that resulted in the discovery of VU6005806/AZN-00016130. While the thieno[2,3- c ]pyridazine core has been a consistent feature of key M 4 PAMs, no work had previously been reported with respect to alternate functionality at the C3 position of the pyridazine ring. Here, we detail new chemistry and analogs that explored this region, and quickly led to VU6005806/AZN-00016130, which was profiled as a putative candidate. While, the β-amino carboxamide moiety engendered solubility limited absorption in higher species precluding advancement (or requiring extensive pharmaceutical sciences formulation), VU6005806/AZN-00016130 represents a new, high quality preclinical in vivo probe. [ABSTRACT FROM AUTHOR]

Highlights • Acetylcholine receptor (AChR) ligands influence astrocyte functions. • ACh suppressed the GFAP expression in differentiation-induced rat glioma C6 cells (differentiated C6 cells). • Carbachol suppressed the GFAP expression in differentiatied C6 cells. • Muscarinic AChR is involved in regulation of the GFAP expression in differentiated C6 cells. Abstract Astrocytes, the most common glial cells in the central nervous system, maintain neuronal functions and have roles in neurological diseases. Acetylcholine (ACh) is one of the most essential neurotransmitters, and ACh receptor (AChR) ligands were recently reported to influence astrocyte functions. However, the functions of ACh, the only endogenous agonist of AChR, in astrocytogenesis and in the expression of astrocytic marker genes have not been known. We previously demonstrated that the inhibition of acetylcholine esterase (AChE) suppressed the differentiation of rat glioma C6 cells, an astrocyte differentiation model, and we observed a suppressive effect of ACh agonists on astrocyte differentiation. Our present study revealed that in the cAMP-induced differentiation of C6 cells, an AChR antagonist alleviated the expression of glia fibrillary acidic protein (GFAP) that had been suppressed by dichlorvos (DDVP), an organophosphate and an AChE inhibitor. Our findings also demonstrated a direct effect of ACh on the GFAP expression, and that muscarinic AChR is involved in the suppressive effect of ACh on the GFAP expression in differentiation-induced C6 cells. This is the first report indicating that ACh the only endogenous agonist for AChRs functions as a mediator of astrocyte differentiation. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • First description of the role of the β-aminocarboxamide moiety within M 4 PAM scaffolds. • Novel des -amino and tricyclic variant M 4 PAMs. • The role of the IMHB motif was established, and could be re-enforced by tricycles. Abstract This letter describes a focused exercise to explore the role of the β-amino carboxamide moiety found in all of the first generation M 4 PAMs and question if the NH 2 group served solely to stabilize an intramolecular hydrogen bond (IMHB) and enforce planarity. To address this issue (and to potentially find a substitute for the β-amino carboxamide that engendered P-gp and contributed to solubility liabilities), we removed the NH 2 , generating des -amino congeners and surveyed other functional groups in the β-position. These modifications led to weak M 4 PAMs with poor DMPK properties. Cyclization of the β-amino carboxamide moiety by virtue of a pyrazole ring re-enforced the IMHB, led to potent (and patented) M 4 PAMs, many as potent as the classical bicyclic β-amino carboxamide analogs, but with significant CYP1A2 inhibition. Overall, this exercise indicated that the β-amino carboxamide moiety most likely facilitates an IMHB, and is essential for M 4 PAM activity within classical bicyclic M 4 PAM scaffolds. [ABSTRACT FROM AUTHOR]

Subtype-selective allosteric modulation of the M 1 muscarinic acetylcholine (ACh) receptor (M 1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M 1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M 1 PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 ( N -((3 R ,4 S )-3-hydroxytetrahydro-2 H -pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)pyridine-2-carboxamide), VU6004256 (4,6-difluoro- N -(1 S ,2 S )-2-hydroxycyclohexyl-1-((6-(1-methyl-1 H -pyrazol-4-yl)pyridine-3-yl)methyl)-1 H -indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1 H -pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3 H )-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M 1 mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W400 7.35 in transmembrane domain (TM) 7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M 1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) have been classically described as monomeric entities that function by binding in a 1:1 stoichiometric ratio to both ligand and downstream signalling proteins. However, in recent years, a growing number of studies has supported the hypothesis that these receptors can interact to form dimers and higher order oligomers although the molecular basis for these interactions, the overall quaternary arrangements and the functional importance of GPCR oligomerization remain topics of intense speculation. Muscarinic acetylcholine receptors belong to class A of the GPCR family. Each muscarinic receptor subtype has its own particular distribution throughout the central and peripheral nervous systems. In the central nervous system, muscarinic receptors regulate several sensory, cognitive, and motor functions while, in the peripheral nervous system, they are involved in the regulation of heart rate, stimulation of glandular secretion and smooth muscle contraction. Muscarinic acetylcholine receptors have long been used as a model for the study of GPCR structure and function and to address aspects of GPCR dimerization using a broad range of approaches. In this review, the prevailing knowledge regarding the quaternary arrangement for the various muscarinic acetylcholine receptors has been summarized by discussing work ranging from initial results obtained using more traditional biochemical approaches to those generated with more modern biophysical techniques. This article is part of the Special Issue entitled ‘Neuropharmacology on Muscarinic Receptors’. [ABSTRACT FROM AUTHOR]

This letter describes the chemical optimization of a new series of M 1 positive allosteric modulators (PAMs) based on a novel benzomorpholine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0486846 ( 7 ), devoid of adverse effect liability. This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0486846 and details all of the challenges faced in allosteric modulator programs (both steep and flat SAR, as well as subtle structural changes affecting CNS penetration and overall physiochemical and DMPK properties). [ABSTRACT FROM AUTHOR]

Herein we describe the continued optimization of M 4 positive allosteric modulators (PAMs) within the 5-amino-thieno[2,3- c ]pyridazine series of compounds. In this letter, we disclose our studies on tertiary amides derived from substituted azetidines. This series provided excellent CNS penetration, which had been challenging to consistently achieve in other amide series. Efforts to mitigate high clearance, aided by metabolic softspot analysis, were unsuccessful and precluded this series from further consideration as a preclinical candidate. In the course of this study, we found that potassium tetrafluoroborate salts could be engaged in a tosyl hydrazone reductive cross coupling reaction, a previously unreported transformation, which expands the synthetic utility of the methodology. [ABSTRACT FROM AUTHOR]

Acetylcholine, as a major neurotransmitter, mediates many brain functions such as pain. This study was aimed to investigate the effects of microinjection of muscarinic and nicotinic acetylcholine receptor antagonists and agonists into the ventrolateral orbital cortex (VLOC) on capsaicin-induced orofacial nociception and subsequent hyperalgesia. The right side of VLOC was surgically implanted with a guide cannula in anaesthetized rats. Orofacial pain-related behaviors were induced by subcutaneous injection of a capsaicin solution (1.5 µg/20 µl) into the left vibrissa pad. The time spent face rubbing with ipsilateral forepaw and general behavior were recorded for 10 min, and then mechanical hyperalgesia was determined using von Frey filaments at 15, 30, 45 and 60 min post-capsaicin injection. Alone intra-VLOC microinjection of atropine (a muscarinic acetylcholine receptor antagonist) and mecamylamine (a nicotinic acetylcholine receptor antagonist) at a similar dose of 200 ng/site did not alter nocifensive behavior and hyperalgesia. Microinjection of oxotremorine (a muscarinic acetylcholine receptor agonist) at doses of 50 and 100 ng/site and epibatidine (a nicotinic acetylcholine receptor agonist) at doses of 12.5, 25, 50 and 100 ng/site into the VLOC suppressed pain-related behaviors. Prior microinjections of 200 ng/site atropine and mecamylamine (200 ng/site) prevented oxotremorine (100 ng/site)-, and epibatidine (100 ng/site)-induced antinociception, respectively. None of the above-mentioned chemicals changed general behavior. These results showed that the VLOC muscarinic and nicotinic acetylcholine receptors might be involved in modulation of orofacial nociception and hypersensitivity. [ABSTRACT FROM AUTHOR]

This Letter details the discovery and subsequent optimization of a novel M 4 PAM scaffold based on an 6-fluoro-4-(piperidin-1-yl)quinoline-3-carbonitrile core, which represents a distinct departure from the classical M 4 PAM chemotypes. Optimized compounds in this series demonstrated improved M 4 PAM potency on both human and rat M 4 (4 to 5-fold relative to HTS hit), and displayed attractive physicochemical and DMPK profiles, including good CNS penetration (rat brain:plasma K p = 5.3, K p,uu = 2.4; MDCK-MDR1 (P-gp) ER = 1.1). [ABSTRACT FROM AUTHOR]

The superior salivatory nucleus (SSN) contains parasympathetic preganglionic neurons innervating the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, is a sialogogue that possibly stimulates SSN neurons in addition to the salivary glands themselves because it can cross the blood-brain barrier (BBB). In the present study, we examined immunoreactivities for mAChR subtypes in SSN neurons retrogradely labeled with a fluorescent tracer in neonatal rats. Additionally, we examined the effects of cevimeline in labeled SSN neurons of brainstem slices using a whole-cell patch-clamp technique. Mainly M1 and M3 receptors were detected by immunohistochemical staining, with low-level detection of M4 and M5 receptors and absence of M2 receptors. Most (110 of 129) SSN neurons exhibited excitatory responses to application of cevimeline. In responding neurons, voltage-clamp recordings showed that 84% (101/120) of the neurons exhibited inward currents. In the neurons displaying inward currents, the effects of the mAChR antagonists were examined. A mixture of M1 and M3 receptor antagonists most effectively reduced the peak amplitude of inward currents, suggesting that the excitatory effects of cevimeline on SSN neurons were mainly mediated by M1 and M3 receptors. Current-clamp recordings showed that application of cevimeline induced membrane depolarization (9/9 neurons). These results suggest that most SSN neurons are excited by cevimeline via M1 and M3 muscarinic receptors. [ABSTRACT FROM AUTHOR]

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M 4 antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M 4 (hM 4 IC 50 s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K p = 2.1, K p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M 1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists. [ABSTRACT FROM AUTHOR]

Intestinal epithelial cells form a tight barrier to act as selective physical barriers, repelling hostile substances. Tumor necrosis factor-α (TNF-α) is a well characterized pro-inflammatory cytokine which can compromise intestinal barrier function and the suppression of TNF-α function is important for treatment of inflammatory bowel disease (IBD). In this study, we investigated the contribution of G-protein-coupled receptor (GPCR)-induced signalling pathways to the maintenance of epithelial barrier function. We first demonstrated the existence of functional muscarinic M3 and histamine H1 receptors in colonic epithelial cell HT-29/B6. As we previously reported, muscarinic M3 receptor prevented TNF-α-induced barrier disruption through acceleration of TNF receptor (TNFR) shedding which is carried out by TNF-α converting enzyme (TACE). M3 receptor-mediated suppression of TNF-α function depends on Gα q/11 protein, however, histamine H1 receptor could not ameliorate TNF-α function, while which could induce Gα q/11 dependent intracellular Ca 2 + mobilization. We found that p38 MAPK was predominantly phosphorylated by M3 receptor through Gα q/11 protein, whereas H1 receptor barely upregulated the phosphorylation. Inhibition of p38 MAPK abolished M3 receptor-mediated TNFR shedding and suppression of TNF-α-induced NF-κB signalling. The p38 MAPK was also involved in TACE- mediated EGFR transactivation followed by ERK1/2 phosphorylation. These results indicate that not H1 but M3 receptor-induced activation of p38 MAPK might contribute to the maintenance of epithelial barrier function through down-regulation of TNF-α signalling and activation of EGFR. [ABSTRACT FROM AUTHOR]

This letter details the continued chemical optimization of a novel series of M 4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3- c ]pyridazine core by incorporating a 3-amino azetidine amide moiety. The analogs described within this work represent the most potent M 4 PAMs reported for this series to date. The SAR to address potency, clearance, subtype selectivity, CNS exposure, and P-gp efflux are described. This work culminated in the discovery of VU6000918, which demonstrated robust efficacy in a rat amphetamine-induced hyperlocomotion reversal model at a minimum efficacious dose of 0.3 mg/kg. [ABSTRACT FROM AUTHOR]

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M 4 antagonists, based on a 4,6-disubstituted core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at both human and rat M 4 (IC 50 s < 300 nM), with no substantial species differences noted. Moreover, CNS penetration proved attractive for this series (brain:plasma K p,uu = 0.87), while other DMPK attributes were addressed in the course of the optimization effort, providing low in vivo clearance in rat (CL p = 5.37 mL/min/kg). Surprisingly, this series displayed pan -muscarinic antagonist activity across M 1–5 , despite the absence of the prototypical basic or quaternary amine moiety, thus offering a new chemotype from which to develop a next generation of pan -muscarinic antagonist agents. [ABSTRACT FROM AUTHOR]

This letter describes the further chemical optimization of the 5-amino-thieno[2,3- c ]pyridazine series (VU0467154/VU0467485) of M 4 positive allosteric modulators (PAMs), developed via iterative parallel synthesis, culminating in the discovery of the non-human primate (NHP) in vivo tool compound, VU0476406 ( 8p ). VU0476406 is an important in vivo tool compound to enable translation of pharmacodynamics from rodent to NHP, and while data related to a Parkinson’s disease model has been reported with 8p , this is the first disclosure of the optimization and discovery of VU0476406, as well as detailed pharmacology and DMPK properties. [ABSTRACT FROM AUTHOR]

This letter describes the continued optimization of M 5 NAM ML375 (VU0483253). While a valuable in vivo tool compound, ML375 has an excessively long elimination half-life in rat (t 1/2 = 80 h), which can be problematic in certain rodent addiction paradigms (e.g., reinstatement). Thus, we required an M 5 NAM of comparable potency to ML375, but with a rat t 1/2 of less than 4 h. Steep SAR plagued this chemotype, and here we detail aniline replacements that offered some improvements over ML375, but failed to advance. Ultimately, incorporation of a single methyl group to the 9 b -phenyl ring acted as a metabolic shunt, providing ( S )- 11 (VU6008667), an equipotent M 5 NAM, with high CNS penetration, excellent selectivity versus M 1–4 and the desired short half-life (t 1/2 = 2.3 h) in rat. [ABSTRACT FROM AUTHOR]

Crack cocaine has a high potential to induce cocaine addiction and its smoke contains cocaine’s pyrolysis product anhydroecgonine methyl ester (AEME), a partial agonist at M 1 - and M 3 -muscarinic acetylcholine receptor and an antagonist at the remaining subtypes. No reports have assessed AEME’s role in addiction. Adult male Wistar rats were intraperitoneally administered with saline, 3 mg/kg AEME, 15 mg/kg cocaine, or a cocaine-AEME combination on every other day during a period of 9 days. After a 7-days withdrawal period, a challenge injection of the respective drugs was performed on the 17th day. The locomotor activity was evaluated on days 1, 3, 5, 7, 9 and 17, as well as dopamine levels (9th day) and dopaminergic receptors proteins (D 1 R and D 2 R on the 17th day) in the caudate-putamen (CPu) and nucleus accumbens (NAc). AEME was not able to induce the expression of behavioral sensitization, but it substantially potentiates cocaine-effects, with cocaine-AEME combination presenting higher expression than cocaine alone. An increase in the dopamine levels in the CPu in all non-saline groups was observed, with the highest levels in the cocaine-AEME group. There was a decrease in D 1 R protein level in this brain region only for cocaine and cocaine-AEME groups. In the NAc, an increase in the dopamine levels was only observed for cocaine and cocaine-AEME groups, with no changes in both D 1 R and D 2 R protein levels. These behavioral and neurochemical data indicate that AEME alone does not elicit behavioral sensitization but it significantly potentiates cocaine effects when co-administered, resulting in dopamine increase in CPu and NAc, brain regions where dopamine release is mediated by cholinergic activity. [ABSTRACT FROM AUTHOR]

Aims Serotonin stimulates enterocyte turnover in the small intestine and studies suggest this is mediated by neuronal signaling via a cholinergic pathway. Distribution of the five known muscarinic receptor subtypes (mAChRs) in the small intestine has not been fully studied, and their role in intestinal growth is unknown. We hypothesized that mAChRs have distinct anatomic distributions within the bowel, and that mAChRs present within intestinal crypts mediate the effects of acetylcholine on the small intestinal mucosa. Main methods Small intestine from male C57BL/6 mice ages 2, 4, 6, and 8 weeks were harvested. RNA was isolated and cDNA synthesized for PCR-amplification of subtype specific mAChRs. Ileum was fixed with Nakane, embedded in epon, and immunofluorescence microscopy performed using polyclonal antibodies specific to each mAChR1-5. Key findings All five mAChR subtypes were present in the mouse duodenum, jejunum, and ileum at all ages by RT-PCR. Immunofluorescence microscopy suggested the presence of mAChR1-5 in association with mature enterocytes along the villus and within the myenteric plexus. Only mAChR2 clearly localized to the crypt stem cell compartment, specifically co-localizing with Paneth cells at crypt bases. Significance Muscarinic receptors are widely distributed along the entire alimentary tract. mAChR2 appears to localize to the crypt stem cell compartment, suggesting it is a plausible regulator of stem cell activity. The location of mAChR2 to the crypt makes it a potential therapeutic target for treatment of intestinal disease such as short bowel syndrome. The exact cellular location and action of each mAChR requires further study. [ABSTRACT FROM AUTHOR]

This letter describes the chemical optimization of a novel series of M 4 positive allosteric modulators (PAMs) based on a 5-amino-thieno[2,3- c ]pyridazine core, developed via iterative parallel synthesis, and culminating in the highly utilized rodent in vivo tool compound, VU0467154 ( 5 ). This is the first report of the optimization campaign (SAR and DMPK profiling) that led to the discovery of VU0467154, and details all of the challenges faced in allosteric modulator programs (steep SAR, species differences in PAM pharmacology and subtle structural changes affecting CNS penetration). [ABSTRACT FROM AUTHOR]

Muscarinic receptors, which selectively take muscarine as their ligand, are critical for the immunological and physiological processes in animals. In the present study, the open region frame (ORF) of a homologue of muscarinic acetylcholine (ACh) receptor (mAChR) was amplified from oyster Crassostrea gigas (named as CgmAChR-1), whose full length was 1983 bp and the protein it encoded contained 660 amino acids with a seven transmembrane region. Phylogeny analysis suggested that CgmAChR-1 shared homology with M 5 muscarinic receptor found in invertebrates including Habropoda laboriosa , Acromyrmex echinatior and Echinococcus granulosus . After cell transfection of CgmAChR-1 into HEK293T cells and ACh incubation, the level of intracellular Ca 2+ and cAMP increased significantly ( p < 0.05). Such trend could be reverted with the addition of M 3 and M 5 muscarinic receptor antagonists DAMP and DAR. The CgmAChR-1 transcripts were ubiquitously detectable in seven different tissues with the maximal expression level in adductor muscle. When the oysters received LPS stimulation, CgmAChR-1 mRNA expression in haemocyte was increased to the highest level (6.05-fold, p < 0.05) at 24 h, while blocking CgmAChR-1 using receptor antagonists before LPS stimulation promoted the expression of oyster TNF, resulting in the increase of haemocyte apoptosis index. These results suggested that CgmAChR-1 was the key molecule in cholinergic neuroendocrine-immune system contributing to the regulation of TNF expression and apoptosis process. [ABSTRACT FROM AUTHOR]

The neuromodulator acetylcholine (ACh) is considered to have a crucial effect on sensory inputs in the process of learning and memory, and ACh activates muscarinic (mAChR) and nicotinic (nAChR) acetylcholine receptors. Meanwhile in a hippocampal CA1 network including inhibitory connections, long-term potentiation (LTP) or long-term depression (LTD) is induced by the application of positive timing of the spike timing-dependent plasticity (STDP) protocol, while LTD is induced by negative timing protocol. In the previous study, the influence of ACh on LTD induced by the negative timing protocol application in the interneuron-blocked CA1 network was reported. However, the responsibility of mAChR and nAChR on pyramidal neuron and interneuron on STDP induction is still unclear. In order to clarify the role of AChRs in LTD, positive or negative timing protocol was applied in the interneuron-activated CA1 network in the presence of eserine. Consequently, the LTD induced by the positive timing protocol was switched to LTP, and the LTD by negative timing protocol was shifted toward potentiation when ACh was effective. The STDP facilitation was more effectively brought by mAChR activation on pyramidal neuron than nAChR, while mAChR on interneuron had a potential to down regulate the facilitation. These findings suggest that the direction (LTD/LTP) of STDP is determined by the activation of mAChR not only on pyramidal neuron but also on interneuron, and the magnitude of STDP is sensitively fine-tuned by nAChR. Therefore, the modulation of synaptic plasticity induced by the coactivation of mAChR and nAChR might be an important stage in integrating ACh and sensory inputs in the hippocampal CA1 network. [ABSTRACT FROM AUTHOR]

Aims T lymphocytes express muscarinic acetylcholine receptors (mAChRs) involved in regulating their proliferation, differentiation and cytokine release. Activation of M 1 , M 3 or M 5 mAChRs increases the intracellular Ca 2 + concentration ([Ca 2 + ] i ) through inositol-1,4,5-phosphate (IP 3 )-mediated Ca 2 + release from endoplasmic reticulum Ca 2 + stores. In addition, T lymphocytes express Ca 2 + -release activated Ca 2 + (CRAC) channels to induce Ca 2 + influx and to regulate diverse immune functions. Our aim in the present study was to assess the role of CRAC channels during mAChR activation in the Ca 2 + -dependent transduction that contributes to the regulation of T cell function. Main methods Changes in [Ca 2 + ] i following mAChR activation on human leukemic T cells, CCRF-CEM (CEM), were monitored using fura-2, based on the ratio of 510 nm fluorescences elicited by excitation at 340 nm and 380 nm ( R 340/380 ). Key findings We demonstrate that CEM cells express mainly M 3 and M 5 mAChRs, but little the M 1 subtype, and that oxotremorine-M (Oxo-M), an mAChR agonist, induces an initial transient increase in [Ca 2 + ] i followed by repetitive [Ca 2 + ] i oscillations. Removing extracellular Ca 2 + or pharmacological blockade of CRAC channels abolished the [Ca 2 + ] i oscillations without affecting the initial [Ca 2 + ] i transient induced by Oxo-M. Moreover, CRAC channel blockade also suppressed Oxo-M-induced c-fos and interleukin-2 expression. Significance These results suggest that upon M 3 or M 5 mAChR activation, IP 3 -mediated Ca 2 + release induces extracellular Ca 2 + influx through CRAC channels, which generates repetitive [Ca 2 + ] i oscillations and, in turn, enhances c-fos gene expression in T lymphocytes. [ABSTRACT FROM AUTHOR]

This Letter describes a ligand-based virtual screening campaign utilizing SAR data around the M 5 NAMs, ML375 and VU6000181. Both QSAR and shape scores were employed to virtually screen a 98,000-member compound library. Neither approach alone proved productive, but a consensus score of the two models identified a novel scaffold which proved to be a modestly selective, but weak inhibitor (VU0549108) of the M 5 mAChR (M 5 IC 50 = 6.2 μM, M 1–4 IC 50 s >10 μM) based on an unusual 8-((1,3,5-trimethyl-1 H -pyrazol-4-yl)sulfonyl)-1-oxa-4-thia-8-azaspiro[4,5]decane scaffold. [ 3 H]-NMS binding studies showed that VU0549108 interacts with the orthosteric site ( K i of 2.7 μM), but it is not clear if this is negative cooperativity or orthosteric binding. Interestingly, analogs synthesized around VU0549108 proved weak, and SAR was very steep. However, this campaign validated the approach and warranted further expansion to identify additional novel chemotypes. [ABSTRACT FROM AUTHOR]

This Letter describes the chemical optimization of a novel series of M 4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M 4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M 4 PAM activity. [ABSTRACT FROM AUTHOR]

It is well known that the cerebellar fastigial nucleus (FN) is involved in cardiovascular modulation, and has direct evidence of cholinergic activity; however, whether and how acetylcholine (ACh) in the FN modulates blood pressure has not been investigated. In this study, we analyzed mean arterial pressure, maximal change in mean arterial pressure, and the reaction time of blood pressure changes after microinjection of cholinergic reagents into the FN in anesthetized rats. The results showed that ACh evoked a concentration-dependent (10, 30 and 100 mM) effect on blood pressure down-regulation. The muscarinic ACh (mACh) receptor antagonist atropine, but not the nicotinic ACh (nACh) receptor antagonist mecamylamine, blocked the ACh-mediated depressor response. The mACh receptor agonist oxotremorine M, rather than nACh receptor agonist nicotine, mimicked the ACh-mediated blood pressure decrease in a dose-dependent manner (10, 30 and 100 mM). These results indicate that cholinergic input in the cerebellar FN exerts a depressor effect on systemic blood pressure regulation, and such effects are substantially contributed by mACh rather than nACh receptors, although the precise mechanism concerning the role of mACh receptor in FN-mediated blood pressure modulation remains to be elucidated. [ABSTRACT FROM AUTHOR]

This Letter describes the continued chemical optimization of the VU0453595 series of M 1 positive allosteric modulators (PAMs). By surveying alternative 5,6- and 6,6-heterobicylic cores for the 6,7-dihydro-5 H -pyrrolo[3,4- b ]pyridine-5-one core of VU453595, we found new cores that engendered not only comparable or improved M 1 PAM potency, but significantly improved CNS distribution ( K p s 0.3–3.1). Moreover, this campaign provided fundamentally distinct M 1 PAM chemotypes, greatly expanding the available structural diversity for this valuable CNS target, devoid of hydrogen-bond donors. [ABSTRACT FROM AUTHOR]

This Letter describes the chemical optimization of a novel series of M 4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3- d ]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma K p = 0.74), within the original core after several rounds of optimization; however, the thieno[2,3- d ]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M 4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma K p > 10). Moreover, this campaign provided fundamentally distinct M 4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target. [ABSTRACT FROM AUTHOR]

A series of homodimers of the well-known cholinergic agonist carbachol have been synthesized, showing the two agonist units symmetrically connected through a methylene chain of variable length. The new compounds have been tested on the five cloned muscarinic receptors (hM 1–5 ) expressed in CHO cells by means of equilibrium binding studies, showing an increase in affinity by rising the number of methylene units up to 7 and 9. Functional experiments on guinea-pig ileum and assessment of ERK1/2 phosphorylation on hM 1 , hM 2 and hM 3 on CHO cells have shown that the new compounds are endowed with muscarinic antagonistic properties. Kinetic binding studies have revealed that some of the tested compounds are able to slow the rate of dissociation of NMS, suggesting a bitopic behavior. Docking simulations, performed on the hM 1 and hM 2 receptors, give a sound rationalization of the experimental data revealing how these compounds are able to interact with both orthosteric and allosteric binding sites depending on the length of their connecting chain. [ABSTRACT FROM AUTHOR]

The decline in spatial working memory is one of the earliest signs of normal brain aging. We developed a novel physical exercise method, termed the "shaking exercise," to slow down this process. The experimental protocol included administering the shaking exercise for 8–32 weeks in male senescence-accelerated mouse prone 10 (SAMP-10). They were subjected to the T-maze test, followed by immunohistochemical analysis, to assess the influence of the shaking exercise on the M1 muscarinic acetylcholine receptor (CHRM1) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) of the dorsal hippocampus and medial prefrontal cortex (dHC-mPFC). The T-maze test demonstrated that the shaking group had less hesitation in the face of selecting direction at week 24. In the immunohistochemical analysis, more CHRM1s were in the CA3 subregion and more AMPARs were in the subiculum. CHRM1s and AMPARs were maintained in the CA1 region and the mPFC. The CHRM1s seem to have a positive effect on the AMPAR in the dentate gyrus (DG) region and the CA3 region. In the CA1 region, CHRM1s were negatively correlated with AMPARs. In addition, high-density neurons were expressed in the shaking group in the upstream DG, the middle part and the distal part of CA3, the distal part of CA1, and the mPFC. Our results raise the possibility that maintenance of the spatial working memory effect observed with the shaking exercise is driven in part by the uneven affection of CHRM1s and AMPARs in the dHC-mPFC circuit system and significantly maintains the neuronal expression in the dHC-mPFC. [Display omitted] • The shaking exercise improved the density of certain neurons involved in memory. • Spatial working memory effect is driven by the uneven activation of CHRM1s and AMPARs. • The shaking exercise was used to keep the function of hippocampus and medial prefrontal cortex. • Shaking exercise-treated SAMP-10 mice maintained spatial working memory longer. [ABSTRACT FROM AUTHOR]

This Letter details our efforts to replace the 3-amino moiety, an essential pharmacophore for M 4 PAM activity in most M 4 PAMs to date, within the thieno[2,3- b ]pyridine core, as the β-amino carboxamide motif has been shown to engender poor solubility, varying degrees of P-gp efflux and represents a structural alert. A scaffold hopping exercise identified a novel 2,4-dimethylquinoline carboxamide core that provided M 4 PAM activity and good CNS penetration without an amino moiety. In addition, MacMillan photoredox catalysis chemistry was essential for construction of the 2,4-dimethylquinoline core. [ABSTRACT FROM AUTHOR]

Anxiety is one of the most prevalent neuropsychiatric disorders, and little is known about its pathogenesis. In order to investigate the neural mechanisms of this mental disorder, we used rat behavior in the elevated plus-maze as an animal model of anxiety and the insular cortex (insula) as a brain target. The microinjection of non-selective and selective M1 and M4 muscarinic acetylcholine receptor (mAChR) agonists or antagonists was used to explore whether the insular muscarinic receptor and its subtypes regulate levels of anxiety. The results showed that both non-selective and selective M1 and M4 mAChR agonists increased the time spent on exploring in the open arms, whereas antagonists decreased exploration. Our results indicate that activation of insular mAChRs could produce anxiolytic effects, whereas inhibition of insular mAChRs could increase anxiety. We concluded that the insular muscarinic system plays a role in the modulation of anxiety, and dysfunction of mAChR signaling may be involved in the mechanism of anxiogenesis. [ABSTRACT FROM AUTHOR]

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [3H]iperoxo - unlike the common radioagonists [3H]acetylcholine and [3H]oxotremorine M - allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [3H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M2 and M4 receptor in the picomolar range. Probing ternary complex formation on the M2 receptor, [3H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M2, the preferred Gi protein acted as a positive allosteric modulator of [3H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM2 cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [3H]iperoxo. Taken together, the muscarinic superagonist [3H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors. [ABSTRACT FROM AUTHOR]

Highlights: [•] Antihypertensive activity of Bj-proline-rich oligopeptides. [•] Diversity of mechanisms for antihypertensive action of Bj-proline-rich oligopeptides. [•] Bj-proline-rich oligopeptides provides synergistic cardiovascular actions through different mechanisms. [•] Complexity of the effects exerted by Bj-PROs for cardiovascular control. [ABSTRACT FROM AUTHOR]

Abstract: We examined ACh-induced [Ca2+]i dynamics in pancreatic acinar cells prepared from mAChR subtype-specific knockout (KO) mice. ACh did not induce any [Ca2+]i increase in the cells isolated from M1/M3 double KO mice. In the cells from M3KO mice, ACh (0.3–3μM) caused a monotonic [Ca2+]i increase. However, we found characteristic oscillatory [Ca2+]i increases in cells from M1KO mice in lower concentrations of ACh (0.03–0.3μM). We investigated the receptor specific pattern of [Ca2+]i increase in COS-7 cells transfected with M1 or M3 receptors. ACh induced the oscillatory [Ca2+]i increase in M3 expressing cells, but not in cells expressing M1, which exhibited monotonic [Ca2+]i increases. IP3 production detected in fluorescent indicator co-transfected cells was higher in M1 than in M3 expressing cells. From the examination of four types of M1/M3 chimera receptors we found that the carboxyl-terminal region of M3 was responsible for the generation of Ca2+ oscillations. The present results suggest that the oscillatory Ca2+ increase in response to M3 stimulation is dependent upon a moderate IP3 increase, which is suitable for causing Ca2+-dependent IP3-induced Ca2+ release. The C-terminal domain of M3 may contribute as a regulator of the efficiency of Gq and PLC cooperation. [Copyright &y& Elsevier]

Abstract: Aims: Recombinant systems have been used for evaluating the properties of G-protein-coupled receptors (GPCRs) on the assumption of cell surface expression. However, many GPCRs, including muscarinic acetylcholine receptors (mAChRs), have also been reported to be distributed in intracellular organelles in native tissues and cell lines. In this study, we compared the pharmacological profiles of exogenously and endogenously expressed M1-mAChRs, and evaluated the functional properties of these receptors. Main methods: Recombinant M1-mAChRs were expressed exogenously in Chinese hamster ovary cells (CHO-M1 cells) and compared with endogenously expressed M1-mAChRs in N1E-115 neuroblastoma cells. The pharmacological and functional profiles were evaluated using cell-permeable antagonists (1-quinuclidinyl-benzilate (QNB), pirenzepine and atropine) and cell-impermeable antagonists (N-methylscopolamine (NMS) or MT-7). Key findings: M1-mAChRs were seen at the cell surface and intracellular sites in both cell lines. Under whole cell conditions, intracellular M1-mAChRs were mainly recognized by cell-permeable ligands, but scarcely by cell-impermeable ligands (at less than 100nM). In CHO-M1 cells, M1-mAChR activation by carbachol resulted in Ca2+ mobilization, ERK1/2 phosphorylation and a reduction in thymidine incorporation, all of which were completely inhibited by MT-7, indicating the involvement of surface M1-mAChRs. In N1E-115 cells, Ca2+ mobilization occurred through surface M1-mAChRs, whereas ERK1/2 phosphorylation and acceleration of thymidine incorporation were mediated through intracellular M1-mAChRs. Significance: Exogenous and endogenous M1-mAChRs are present at both the cell surface and the intracellular organelles, and the pharmacological properties of geographically distinct M1-mAChRs are different, and may depend on cell background and/or exogenous or endogenous origin. [Copyright &y& Elsevier]

Highlights: [•] The sire's retrieval behavior is inhibited by scopolamine. [•] The sire's retrieval parental behavior is inhibited by retigabine. [•] Mouse male's parental behavior is sensitive to the central cholinergic signaling. [ABSTRACT FROM AUTHOR]

Abstract: Aims: We have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. Main methods: This small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. Key findings: The MT-Mlα was able to displace the [3H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [3H]QNB-binding site with pKi values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M1 antagonist pirenzepine caused a dose-dependent block on total [3H]inositol phosphate accumulation induced by carbachol. The IC50 values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. Significance: The results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs. [Copyright &y& Elsevier]

Abstract: In previous work, we have shown that light-adaptive pigment granule dispersion can be induced in vitro by treating retinal pigment epithelium (RPE) isolated from bluegill retina with acetylcholine or its analog carbachol and that these agents act through muscarinic receptors to induce pigment granule dispersion. RPE is a monolayer of tissue found between the neural retina and the choroid. In fish, RPE has long apical projections enmeshed with the distal part of photoreceptors, reaching down to the level of their nuclei. The RPE disperses melanin pigment granules into the apical projections to shield light-sensitive photoreceptor outer segments from photobleaching when fish are under bright-light conditions. During development, RPE begin to respond to light at 5days post-fertilization, raising the question of whether responsiveness is correlated to receptor expression. Here, we isolate, clone and sequence chrm-odd receptor genes in zebrafish, characterize them phylogenetically and observe their expression in the eyes of the zebrafish at different developmental stages using RT-PCR and immunofluorescence microscopy. We find that zebrafish express six unique chrm-odd receptor subtypes: chrm1a, chrm1b, chrm3a, chrm3b, chrm5a and chrm5b — and these receptors are differentially expressed during development. Our phylogenetic analysis confirms the assignments of chrm1b and chrm5b, isolated here, as well as other muscarinic receptor genes and their duplicates and suggests previously described muscarinic receptors may need to be reclassified. Differences between the expression patterns of ostensibly duplicated genes raise the possibility that subtle differences between the duplicates may enable refined regulation of specific developmental events. [Copyright &y& Elsevier]

Abstract: The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood–brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors. [Copyright &y& Elsevier]

Abstract: Bradykinin-potentiating peptides (BPPs) or proline-rich oligopeptides (PROs) isolated from the venom glands of Bothrops jararaca (Bj) were the first natural inhibitors of the angiotensin-converting enzyme (ACE) described. Bj-PRO-5a (

Abstract: Acetylcholine is the predominant neurotransmitter in the neuromuscular junction, and a role in bone has been postulated. The expression of nicotinic receptors has been reported in osteoblasts, but the expression and function of muscarinic receptor in bone remain obscure. In this study, we investigated the expression and functional activities of muscarinic receptor subtypes in human osteoblast cell lines and animal and human bone tissue. The mRNA levels of muscarinic receptor subtypes were detected by reverse-transcription polymerase chain reaction. We found that muscarinic subtypes m1, m2, m3, m4, and m5 were expressed at different levels in human osteosarcoma HOS cells, rat femur, and human rib bone tissue; m1, m4, m5 were in cultured mouse femur bone cells and cultured mouse calvarial bone cells; m2, m3, m4 were in bovine bone. The mRNA of neuronal markers, light-, medium- and heavy-neurofilament, was not found in human bone tissues to exclude the possible contamination from neuronal tissue. Methacholine induced an elevation in cytosolic calcium concentration and proliferation in HOS cells. Both effects were blocked by atropine. We conclude that muscarinic receptor is present in bone tissue to evoke calcium signaling and modulate cell proliferation. Different muscarinic receptor subtypes are distributed in various parts of the animal skeletal system including the different species and bone portions. Bone remodeling involving osteoblast proliferation leads the possibilities that muscarinic receptor may play roles in bone remodeling. [ABSTRACT FROM AUTHOR]