M1 Receptor Subtypes Research Articles

A golden age of muscarinic acetylcholine receptor modulation in neurological diseases.

Over the past 40 years, the muscarinic acetylcholine receptor family, particularly the M1-receptor and M4-receptor subtypes, have emerged as validated targets for the symptomatic treatment of neurological diseases such as schizophrenia and Alzheimer disease. However, despite considerable effort and investment, no drugs have yet gained clinical approval. This is largely attributable to cholinergic adverse effects that have halted the majority of programmes and resulted in a waning of interest in these G-protein-coupled receptor targets. Recently, this trend has been reversed. Driven by advances in structure-based drug design and an appreciation of the optimal pharmacological properties necessary to deliver clinical efficacy while minimizing adverse effects, a new generation of M1-receptor and M4-receptor orthosteric agonists and positive allosteric modulators are now entering the clinic. These agents offer the prospect of novel therapeutic solutions for 'hard to treat' neurological diseases, heralding a new era of muscarinic drug discovery.

Distribution of muscarinic acetylcholine receptors in hypoglossal motoneurons across postnatal maturation

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. During REM sleep, hypoglossal motoneurons (XII MNs), which innervate genioglossus, receive less excitatory noradrenergic drive and may be inhibited by activation of muscarinic acetylcholine receptors. Data from intact adult rats during natural sleep implicates an inhibitory effect of muscarinic acetylcholine receptors on XII MNs. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at XII MNs. Our project examines the changes to distribution of muscarinic acetylcholine receptors (mAChRs) at XII MNs across postnatal maturation. We hypothesize there is an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) mAChRs at XII MNs with postnatal maturation. We performed double-labeled immunofluorescence experiments on perfused transverse brainstem slices (20μm) under identical conditions across six postnatal age groups: P0-P2, P3-P5, P6-P10, P11-P13, P14-P17, and adult against mAChR targets M1, M2, M3, M5. Slices were co-labeled with choline acetyltransferase (ChAT) and 4’,6-diamidino-2-phenylindole (DAPI). Images were collected using a confocal microscope. 10 individual XII MNs were identified in each slice using positive ChAT labeling as well as anatomical location. ImageJ was used to determine the average XII MN mAChR subtype intensity. Preliminary data (n = 3) indicate that M1 receptors showed a transitory increase in labeling intensity in XII MNs followed by a decrease across development (P1=89±11%, P4-5=100%, P9=76±18%, P12-13=67±8%, P17=67±4%, adult=56±7%, p = 0.13). M3 (n = 3) showed a trend for a transient increase in labeling intensity at P17 (P1=78±41%, P4-5=87±3%, P9=61±13%, P12-13=86±34%, P17=100%, adult=80±23, p = 0.60). M5 (n = 3) expression intensity decreased consistently over maturation (P1=100%, P4-5=92±34%, P9=75±23%, P12-13=70±10%, P17=66±14%, adult=39±12%, p = 0.0257). In contrast, M2 (n = 3) receptor expression intensity remained relatively consistent across maturation (P1 = 88±23%, P4-5 = 100%, P9=88±10%, P12-13 = 75±15%, P17 = 86±18%, adult = 75±10%). These data partly support our hypothesis of a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors across maturation. The decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift in muscarinic modulation from excitation to inhibition with postnatal maturation. Arizona Alzheimer's Consortium, NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

M3 Receptor Pathway Stimulates Rapid Transcription of the CB1 Receptor Activation through Calcium Signalling and the CNR1 Gene Promoter.

In this study, we have investigated a possible mechanism that enables CB1/M3 receptor cross-talk, using SH-SY5Y cells as a model system. Our results show that M3 receptor activation initiates signaling that rapidly upregulates the CNR1 gene, resulting in a greatly potentiated CB1 receptor response to agonists. Calcium homeostasis plays an essential intermediary role in this functional CB1/M3 receptor cross-talk. We show that M3 receptor-triggered calcium release greatly increases CB1 receptor expression via both transcriptional and translational activity, by enhancing CNR1 promoter activity. The co-expression of M3 and CB1 receptors in brain areas such as the nucleus accumbens and amygdala support the hypothesis that the altered synaptic plasticity observed after exposure to cannabinoids involves cross-talk with the M3 receptor subtype. In this context, M3 receptors and their interaction with the cannabinoid system at the transcriptional level represent a potential pharmacogenomic target not only for the develop of new drugs for addressing addiction and tolerance. but also to understand the mechanisms underpinning response stratification to cannabinoids.

Risk of delirium associated with antimuscarinics in older adults: A case-time-control study.

BackgroundOlder adults are at an increased risk of delirium because of age, polypharmacy, multiple comorbidities and acute illness. Antimuscarinics are the backbone of the pharmacological management of overactive bladder. However, the safety profiles of antimuscarinics vary because of their dissimilarities to muscarinic receptor‐subtype affinities and are associated with differential central anticholinergic adverse effects.ObjectiveThis study aimed to examine delirium risk in new users of oxybutynin and solifenacin in older adults (≥ 65 years). In the secondary analyses, we examined the risk of delirium by type and dose of antimuscarinic.MethodWe applied a case‐time‐control design to investigate delirium risk in older adults who started taking oxybutynin and solifenacin. We used a nationwide inpatient hospital data (2005–2016), National Minimum Data Set, maintained by the Ministry of Health, New Zealand (NZ), to identify older adults with a new‐onset diagnosis of delirium. Eligible patients were older adults aged 65 at entry into the cohort on 1/1/2006. We used dispensing claims data to determine antimuscarinic treatment exposure. The antimuscarinic included in the study were new users of oxybutynin and solifenacin. These two antimuscarinics are subsidised by the Pharmaceutical Management Agency and are the most frequently used antimuscarinic in NZ. A conditional logistic regression model was used to compute matched odds ratios (MORs) and 95% confidence intervals (CIs). In the case‐time‐control design, we made separate analyses to evaluate the dose–response risk of delirium.ResultsWe identified 4818 individuals (mean age 82.14) from 2005 to 2015 with incident delirium and were exposed to at least one of the antimuscarinic of interest. The case‐time‐control matched odds ratio (MOR) for delirium with oxybutynin was (2.06, 95% confidence interval [CI] 1.07–3.96). Solifenacin was not associated with delirium (0.89 95%CI 0.64–1.23). In the sensitivity analyses, the case‐time‐control MOR for delirium using a shorter risk period (0–3 days) did not change the results. The dose–response risk of delirium was significant for oxybutynin (0.05, 95%CI 0.02–0.08) but not for solifenacin (−0.01, 95%CI −0.03 to 0.00). In addition, in the subgroup analyses, a statistically significant association of delirium was found for oxybutynin but not for solifenacin in the non‐dementia cohort (2.11,95% CI 1.08–4.13) and the dementia cohort (1.25, 95%CI 0.05–26.9).ConclusionThe study found that oxybutynin but not solifenacin is associated with a risk of new‐onset delirium in older adults. The higher blockade of M1 and M2 receptors by oxybutynin is likely to contribute to delirium than solifenacin, which is highly selective for the M3 receptor subtype. Therefore, the treatment choice with an M3 selective agent must be given due consideration, particularly in those with pre‐existing cognitive impairment.

Inactivation of the cholinergic M4 receptor results in a disinhibited endophenotype predicting alcohol use

The muscarinic cholinergic M4 receptor subtype (M4 mAChR) is densely expressed in brain areas known to be involved in the reinforcing effects of drugs of abuse and we were the first to show that mice lacking M4 mAChRs exhibit elevated operant responding for alcohol and reduced capacity to extinguish this alcohol-seeking behaviour. Here we explore possible underlying determinants of this phenotype. We subjected M4 mAChR knockout mice and their littermate wildtype controls to tests of spontaneous activity, learning and memory, novelty seeking, as well as anxiety and examined the relationship of a newly discovered “disinhibited” endophenotype of these mice with voluntary alcohol consumption and relapse. We found a positive correlation between “disinhibited” behaviour on the plus maze and alcohol preference as well as relapse to alcohol drinking after a period of abstinence.Taken together, these data point to M4 mAChRs as a potential target for improved treatment strategies for alcohol use disorder. This receptor should be further investigated for its involvement in modulating behavioural inhibition in relation to loss of control over consumption of alcohol.

Effects of postnatal maturation on muscarinic acetylcholine receptor distribution in hypoglossal motorneurons

Upper airway patency is decreased during rapid eye movement (REM) sleep due to loss of genioglossus (primary tongue protruder) tongue muscle tone. Hypoglossal motoneurons (XII MNs), which innervate the genioglossus muscle, receive less excitatory noradrenergic drive, and may be inhibited by activation of muscarinic acetylcholine receptors, during REM sleep. Indeed, preliminary data from intact adult rats during natural sleep implicated an inhibitory effect of muscarinic acetylcholine receptors on hypoglossal motoneurons. However, data using the rhythmic slice preparation from neonatal mice indicate that muscarine has a net excitatory effect on inspiratory burst amplitude at hypoglossal motoneurons. Our project examines the distribution of muscarinic acetylcholine receptors at XII MNs, and how that changes across postnatal maturation. We hypothesizes there would be an increase in inhibitory (M2) and decrease in excitatory (M1, M3, M5) muscarinic receptors in XII MNs with postnatal maturation. We performed double‐labeled immunofluorescence experiments on perfused 20μm transverse brainstem slices across six postnatal age groups under identical conditions: P0‐P2, P3‐P5, P6‐P10, P11‐P13, P14‐P17, and adult against muscarinic targets M1, M2, M3, M5. Slices were co‐labeled with choline acetyltransferase (ChAT) and 4’,6‐diamidino‐2‐phenylindole (DAPI). Images of the hypoglossal motor nucleus (HMN) were collected using a confocal microscope. ImageJ was used to determine the average HMN muscarine receptor subtype intensity across postnatal maturation. Preliminary data (n = 1) indicate that M1 receptors showed a consistent decrease in labeling intensity in the HMN across development (P5=100%, P13=80%, P17=67%, adult = 40%), whereas M3 (n = 1) (P4= 90%, P9= 73%, P13=89%, P17= 100%, adult= 41%) showed a transitory increases in expression before a final decrease into adulthood. M5 (n = 1 or 2) expression shows a bimodal distribution of labeling intensity (P0‐2= 100%, P4‐6= 58‐99%, P7‐10 = 52‐70%, P11‐13= 35‐58 %, P14‐17 = 27 % adult= 17‐37 %). In contrast, M2 receptor expression intensity remained relatively high with small fluctuations into adulthood (n=2), (P0‐2 = 92‐94 %, P4‐6 = 100%, P7‐10 = 92‐95 %, P11‐13 = 78‐107 %, P14‐17 = 88‐107 %, adult = 73‐77 %). These data partly support our hypothesis that there would be a decrease in expression intensity of excitatory M1, M3, and M5 receptor subtypes into adulthood. Contrary to our hypothesis, we observed minimal change in the expression intensity of inhibitory M2 receptors. We speculate that the decrease in labeling intensity of excitatory muscarinic receptor subtypes may support the observed shift of muscarinic modulation from excitation to inhibition with postnatal maturation.

Understanding Why Muscarinic Receptor Agonists Have Antipsychotic Properties

All current antipsychotics have direct dopamine (DA) D2 receptor activity, which is associated with problems such as dysphoria, EPS, or prolactin elevation. Muscarinic receptor agonists have shown antipsychotic-like activity across preclinical models and clinical trials. This poster reviews preclinical evidence as to how muscarinic receptor agonists, such as the M1/M4 preferring agonist xanomeline, might have clinically relevant antipsychotic effects. Highlight the novel mechanisms through which muscarinic receptor agonists are associated with antipsychotic effects without having any direct dopaminergic D2 receptor activity. The muscarinic receptor family is composed of 5 G protein-coupled receptors (GPCRs). One of the leading hypotheses explaining the antipsychotic activity of muscarinic receptor agonists is preclinical studies of muscarinic receptor modulation of those DA circuits associated with psychosis. Both M1 and M4 receptors are expressed in DA neural circuits implicated in psychosis, and provide unique regulation of these circuits. Xanomeline has both functional M1 and M4 receptor agonist activity, and shows robust antipsychotic-like effects in several animal models that require the presence of functional M1 and M4 receptors. M4 receptors are autoreceptors on cholinergic neurons that regulate DA circuits in two locations: ventral tegmental area (VTA) and nucleus accumbens (NAc). Cholinergic-rich neurons from the hindbrain at the VTA, where they release acetylcholine (ACh) into DA-rich synaptic spaces. M4 autoreceptors are present on these neurons, and their activation reduces ACh release and lowers ambient synaptic ACh concentrations, leading to reduced DA cell firing. Cholinergic interneurons residing in the NAc also express M4 autoreceptors. These ACh interneurons regulate ACh release with M4 activation also turning off ACh release. Therefore, M4 receptors serve as DA regulators at VTA and NAc, both key sites for dopamine's role in psychotic processes. M1 receptors regulate DA circuits in a different, "top down" manner. M1 receptors are found on cortical inhibitory interneurons. When activated, inhibitory drive onto excitatory output neurons is enhanced, which leads to reduced excitatory tone to VTA DA neurons. Over the last 25years, a growing body of evidence has shown potential for muscarinic receptor agonists to become a new class of medicines with potent antipsychotic activity. Preclinical data at micro-and macro-circuit levels suggest that the M1 and M4 receptor subtypes are most relevant in the regulation of DA circuits. The antipsychotic effects of muscarinic agonists effects may arise from influencing these key muscarinic receptor subtypes that are integral to the regulation of DA neural circuits. In summary, there has been great progress in understanding the potential for muscarinic receptor agonists for the treatment of psychosis. Karuna Therapeutics.

Subtype-selective contribution of muscarinic acetylcholine receptors for filial imprinting in newly-hatched domestic chicks

Muscarinic acetylcholine receptors (mAChRs) play an important role in many brain functions. Our previous study revealed that the injection of mAChRs antagonist scopolamine into the intermediate medial mesopallium (IMM) region, which is critical for filial imprinting, impairs memory formation. In avian brains, four mAChR subtypes have been identified (M2, M3, M4 and M5). M3 and M5 receptors increase the excitability of neurons, whereas M2 and M4 receptors reduce the excitability. Because the scopolamine blocks all subtypes, the previous study did not identify which subtype contributes to the memory formation. By injecting several types of mAChR antagonists into the IMM, in this study we determined which mAChR subtype plays a critical role in imprinting. First, the effects of antagonists on the excitatory receptor subtypes M3 and M5 were examined. Injection of the M3 antagonist (DAU5884) at 20 mM or the M5 antagonist (ML381) at 2 mM impaired imprinting. Considering the pKi value of DAU5884, the impairment seems to be caused by DAU5884 binding to M3 and/or M4 receptors. Second, the effect of antagonists on the inhibitory receptor subtype M2 was examined. The results showed that the M2 antagonist (AQ-RA741) impaired imprinting at a concentration of 20 mM. Considering the pKi value of AQ-RA741, the impairment seems to be caused by AQ-RA741 binding to M2 and/or M4. The findings of this study suggests that the excitatory receptor subtypes M3 and M5 and the inhibitory receptor subtype M2 and/or M4 cooperate to achieve the appropriate balance of acetylcholine signaling to execute imprinting.

Notch Signal Mediates the Cross-Interaction between M2 Muscarinic Acetylcholine Receptor and Neuregulin/ErbB Pathway: Effects on Schwann Cell Proliferation.

The cross-talk between axon and glial cells during development and in adulthood is mediated by several molecules. Among them are neurotransmitters and their receptors, which are involved in the control of myelinating and non-myelinating glial cell development and physiology. Our previous studies largely demonstrate the functional expression of cholinergic muscarinic receptors in Schwann cells. In particular, the M2 muscarinic receptor subtype, the most abundant cholinergic receptor expressed in Schwann cells, inhibits cell proliferation downregulating proteins expressed in the immature phenotype and triggers promyelinating differentiation genes. In this study, we analysed the in vitro modulation of the Neuregulin-1 (NRG1)/erbB pathway, mediated by the M2 receptor activation, through the selective agonist arecaidine propargyl ester (APE). M2 agonist treatment significantly downregulates NRG1 and erbB receptors expression, both at transcriptional and protein level, and causes the internalization and intracellular accumulation of the erbB2 receptor. Additionally, starting from our previous results concerning the negative modulation of Notch-active fragment NICD by M2 receptor activation, in this work, we clearly demonstrate that the M2 receptor subtype inhibits erbB2 receptors by Notch-1/NICD downregulation. Our data, together with our previous results, demonstrate the existence of a cross-interaction between the M2 receptor and NRG1/erbB pathway-Notch1 mediated, and that it is responsible for the modulation of Schwann cell proliferation/differentiation.

Evaluation of the effects of donepezil, memantine and α-lipoic acid combined administration in amnesia rats on impaired cognitive functions in terms of behavioural, apoptotic, cholinergic and glutamatergic systems

Objective: The aim of the study was to evaluate the possible protective effects of alpha-lipoic acid (α-LA), donepezil and memantine combined therapy in the scopolamine-induced amnesia rat model. 
 Objective: The aim of the study was to evaluate the possible protective effect of donepezil, memantine and alpha-lipoic acid (α-LA) combined therapy in the scopolamine-induced amnesia rat model. 
 Methods: In this study, the effect of combined therapy used in the treatment of scopolamine-induced amnesia on behavioural parameters was evaluated using Y-maze and new object recognition (NOR) test. In addition, muscarinic acetylcholine receptor subtype M1, N-methyl-D-aspartate receptor NR2B subunit, brain-derived neurotrophic factor (BDNF) and mitochondrial apoptosis-related proteins [B-cell lymphoma-2 (Bcl-2) / Bcl-2 associated X (Bax) ratio, caspase (casp) -3, and -9] expression levels were evaluated using the western blot method in the frontal cortex and hippocampus regions. 
 Results: The main findings of this study demonstrated that in scopolamine-induced amnesia rats, cognitive dysfunction determined by both the Y-maze and the NOR test were reversed with the combined treatment of memantine, donepezil and α-LA. According to immunoblotting results in both brain regions, scopolamine-induced decreased M1, BDNF, Bcl-2 / Bax ratio and increased NR2B, casp-3 and -9 expression levels were found to be reversed to almost control values with combined treatment. 
 Conclusion: Consistent with the literature, our study results suggest that the positive contribution of α-LA to the combined treatment of donepezil and memantine, which is used in the routine treatment of neurodegenerative diseases, may be a treatment option in the future.
 Results: The main findings of this study demonstrated that in scopolamine-induced amnesia rats, cognitive dysfunction determined by both the Y-maze and the NORT test were reversed with the combined treatment of memantine, donepezil and α-LA. In addition, decreased BDNF, M1, bcl-2/bax ratio and increased NR2B, caspase-3 and caspase-9 expression levels due to scopolamine in both regions were improved with α-LA, donepezil and memantine combined therapy. 
 Conclusion: The results of our study and other study results in the literature suggest that α-LA may be a future treatment option due to its positive contribution to the effects of other drugs used in the routine treatment of neurodegenerative diseases.

Aged Mice Devoid of the M3 Muscarinic Acetylcholine Receptor Develop Mild Dry Eye Disease.

The parasympathetic nervous system is critically involved in the regulation of tear secretion by activating muscarinic acetylcholine receptors. Hence, various animal models targeting parasympathetic signaling have been developed to induce dry eye disease (DED). However, the muscarinic receptor subtype (M1–M5) mediating tear secretion remains to be determined. This study was conducted to test the hypothesis that the M3 receptor subtype regulates tear secretion and to evaluate the ocular surface phenotype of mice with targeted disruption of the M3 receptor (M3R−/−). The experimental techniques included quantification of tear production, fluorescein staining of the ocular surface, environmental scanning electron microscopy, assessment of proliferating cells in the corneal epithelium and of goblet cells in the conjunctiva, quantification of mRNA for inflammatory cytokines and prooxidant redox enzymes and quantification of reactive oxygen species. Tear volume was reduced in M3R−/− mice compared to age-matched controls at the age of 3 months and 15 months, respectively. This was associated with mild corneal epitheliopathy in the 15-month-old but not in the 3-month-old M3R−/− mice. M3R−/− mice at the age of 15 months also displayed changes in corneal epithelial cell texture, reduced conjunctival goblet cell density, oxidative stress and elevated mRNA expression levels for inflammatory cytokines and prooxidant redox enzymes. The findings suggest that the M3 receptor plays a pivotal role in tear production and its absence leads to ocular surface changes typical for DED at advanced age.

Cholinergic Modulation by Activation of Muscarinic Acetylcholine Receptor Subtypes Modulates Inspiratory Bursting in Hypoglossal Motoneurons in Mice in Vitro

During wakefulness, tonic and respiratory input provides drive to upper airway dilatory muscles, and in particular genioglossus, the main tongue protruder, which stiffens the airway and keeps airway resistance low. These respiratory muscles experience decrements in activity during rapid eye movement sleep. Disruption in motor output and insufficient muscle tone of the tongue due to inhibition, or loss of excitation, of hypoglossal motoneurons (which innervate tongue muscles) contributes to sleep-disordered breathing. Recent data from adult rats in vivo suggest that muscarinic inhibition of hypoglossal motoneurons may be a major mechanism leading to loss of tongue muscle tone during sleep. However, available data indicate that there are contrasting effects of muscarinic modulation: in neonatal rhythmic slice preparations, muscarine applied within the hypoglossal nucleus has an excitatory effect on inspiratory burst amplitude. Muscarinic receptor subtypes can be broadly divided into those that are coupled to excitatory G-protein coupled receptors (M1, M3, M5) and those that are coupled to inhibitory G-protein coupled receptors (M2, M4). Hypoglossal motoneurons express M1, M2, M5, and possibly M3 receptor subtypes. Many modulatory systems undergo postnatal maturation, which we hypothesize underlies the switch in muscarinic modulation from net excitation to net inhibition of inspiratory bursting behavior at hypoglossal motoneurons. As an initial test of our hypothesis, our goal was to determine the contribution of excitatory and inhibitory muscarine receptor subtypes to the muscarinic modulation of inspiratory burst output from hypoglossal motoneurons in neonatal mice. Using the in vitro rhythmic slice preparation, we first investigated inspiratory behavior by observing the net effects of muscarine locally applied into the hypoglossal nucleus (100 mM, 30 s) on inspiratory burst amplitude in neonatal CD-1 mice (postnatal day, P0-P5). Our data confirm that in mice ages P0-5, local application of muscarine increased inspiratory burst amplitude (range: 53-180% potentiation, n = 9) compared to control inspiratory burst activity. Preliminary results suggest that the local application of M2 antagonist methoctramine in addition to muscarine (range: 20%-135% potentiation, n=4) had little effect on inspiratory behavior compared to baseline effects of muscarine (range: 4%-143% potentiation). Next, we tested the contribution of M3 receptors to muscarinic modulation on inspiratory burst amplitude. At baseline, locally applied muscarine increased inspiratory burst amplitude (range: 20%-63% potentiation); the muscarinic potentiation was attenuated during bath application of the M3 receptor-preferring antagonist 4-DAMP (range: 17%-25% potentiation) and recovered upon washout (range: 13%-117% potentiation, n=3). These preliminary results indicate muscarinic excitation is mediated in part by the M3-receptor subtype. Future experiments will determine how the M2 and M3 receptor contributions change with postnatal maturation.

Expression of muscarinic acetylcholine receptors in turkey cardiac chambers

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

Targeting muscarinic receptors to treat schizophrenia

Schizophrenia is a severe neuropsychiatric disorder characterized by a diverse range of symptoms that can have profound impacts on the lives of patients. Currently available antipsychotics target dopamine receptors, and while they are useful for ameliorating the positive symptoms of the disorder, this approach often does not significantly improve negative and cognitive symptoms. Excitingly, preclinical and clinical research suggests that targeting specific muscarinic acetylcholine receptor subtypes could provide more comprehensive symptomatic relief with the potential to ameliorate numerous symptom domains. Mechanistic studies reveal that M1, M4, and M5 receptor subtypes can modulate the specific brain circuits and physiology that are disrupted in schizophrenia and are thought to underlie positive, negative, and cognitive symptoms. Novel therapeutic strategies for targeting these receptors are now advancing in clinical and preclinical development and expand upon the promise of these new treatment strategies to potentially provide more comprehensive relief than currently available antipsychotics.

Design, Formulation and In Vitro Evaluation of Oxybutynin HCl Floating Matrix Tablets

The goal of this study was to prepare and test a hydrodynamically balanced floating matrix controlled Oxybutynin HCl drug delivery system. The muscarinic acetylcholine receptor subtypes M1, M2, and M3 are competitively antagonized by oxybutynin HCl. It is used to treat urinary and bladder problems. It has a 10-12 h elimination half-life. The effervescence produced by the combination of sodium bicarbonate with hydrochloric acid in the stomach causes the Oxybutynin HCl tablets to float in the stomach. Twelve alternative floating tablet formulations were made utilizing direct compression using hydrophilic polymers like HPMC K4M, K15M, and K100M and hydrophobic polymers such ethyl cellulose in various ratios. The produced formulation was characterized using FTIR and DSC analysis. In terms of general appearance, content consistency, hardness, friability, and buoyancy, the examination found that all formulations meet the specifications of official pharmacopoeias and/or standard reference. Formulation F11, which contained 25% HPMC K100M and 12.5% ethyl cellulose, had the best in vitro drug release up to 99% after 12 hours. The formulations with more than 12.5% NaHCO3 had a floating time of more than 12 hours. In vitro drug release kinetics of improved formulation F11 were discovered to be zero order, with anomalous diffusion coupled with erosion being the drug release mechanism. At the conclusion of 90 days, accelerated stability studies revealed negligible change in physicochemical attributes and drug release profiles, indicating that all the formulations were stable.

The metronomic combination of paclitaxel with cholinergic agonists inhibits triple negative breast tumor progression. Participation of M2 receptor subtype.

Triple negative tumors are more aggressive than other breast cancer subtypes and there is a lack of specific therapeutic targets on them. Since muscarinic receptors have been linked to tumor progression, we investigated the effect of metronomic therapy employing a traditional anti-cancer drug, paclitaxel plus muscarinic agonists at low doses on this type of tumor. We observed that MDA-MB231 tumor cells express muscarinic receptors, while they are absent in the non-tumorigenic MCF-10A cell line, which was used as control. The addition of carbachol or arecaidine propargyl ester, a non-selective or a selective subtype 2 muscarinic receptor agonist respectively, plus paclitaxel reduces cell viability involving a down-regulation in the expression of ATP "binding cassette" G2 drug transporter and epidermal growth factor receptor. We also detected an inhibition of tumor cell migration and anti-angiogenic effects produced by those drug combinations in vitro and in vivo (in NUDE mice) respectively. Our findings provide substantial evidence about subtype 2 muscarinic receptors as therapeutic targets for the treatment of triple negative tumors.

First-in-Human Assessment of 11C-LSN3172176, an M1 Muscarinic Acetylcholine Receptor PET Radiotracer.

This was a first-in-human study of the PET radiotracer 11C-LSN3172176 for the muscarinic acetylcholine receptor subtype M1. The objectives of the study were to determine the appropriate kinetic model to quantify binding of the tracer to M1 receptors, and the reliability of the chosen quantification method. Methods: Six healthy subjects completed the test-retest protocol, and 5 healthy subjects completed the baseline-scopolamine blocking protocol. Multiple modeling methods were applied to calculate total distribution volume (VT) and nondisplaceable binding potential (BPND) in various brain regions. The reference region was selected from the blocking study. The occupancy plot was applied to compute receptor occupancy by scopolamine and nondisplaceable distribution volume. Results: Tracer uptake was highest in the striatum, followed by neocortical regions and white matter, and lowest in the cerebellum. Regional time-activity curves were fitted well by all models. The 2-tissue-compartment (2TC) model fits were good, but the 2TC parameters often could not be reliably estimated. Because VT correlated well between the 2TC and 1-tissue-compartment (1TC) models after exclusion of unreliable estimates, the 1TC model was chosen as the most appropriate. The cerebellum showed the lowest VT, consistent with preclinical studies showing little to no specific binding in the region. Further, cerebellar VT did not change between baseline and blocking scans, indicating that the cerebellum is a suitable reference region. The simplified reference tissue model (SRTM) slightly underestimated 1TC BPND, and the simplified reference tissue model 2 (SRTM2) improved BPND estimation. An 80-min scan was sufficient to quantify VT and BPND The test-retest study showed excellent absolute test-retest variability for 1TC VT (≤5%) and BPND (≤10%). In the baseline and blocking studies, occupancy values were lower in the striatum than in nonstriatal regions, as may be attributed to differences in regional acetylcholine concentrations. Conclusion: The 1TC and SRTM2 models are appropriate for quantitative analysis of 11C-LSN3172176 imaging data. 11C-LSN3172176 displayed excellent test-retest reproducibility and is a highly promising ligand to quantify M1 receptors in the human brain.

The M1 muscarinic acetylcholine receptor in the crypt stem cell compartment mediates intestinal mucosal growth.

Localization of a specific subtype of the muscarinic acetylcholine receptor in the crypt stem cell compartment suggests a critical role in intestinal mucosal homeostasis. Here we demonstrate the localization of the M1 muscarinic acetylcholine receptor to the stem cell compartment and demonstrate increase morphometric and proliferative parameters when this is stimulated in vivo. These data provide novel information about this complex signaling microenvironment and offer potential future therapeutic targets for future study.

A Structural Framework for the G Protein Selectivity of Muscarinic Acetylcholine GPCRs

The neurotransmitter molecule acetylcholine is capable of activating five muscarinic acetylcholine receptors, M1 through M5, which belong to the superfamily of G‐protein‐coupled receptors (GPCRs). These five receptors share high sequence and structure homology; however, the M1, M3, and M5 receptor subtypes signal preferentially through the Gαq/11 subset of G proteins, whereas the M2 and M4 receptor subtypes signal through the Gαi/o subset of G proteins, resulting in very different intracellular signaling cascades and physiological effects. The structural basis for this innate ability of the M1/M3/M5 set of receptors and the highly homologous M2/M4 set of receptors to couple to different G proteins is poorly understood. In this study, we used molecular dynamics (MD) simulations coupled with thermodynamic analyses of M1 and M2 receptors coupled to both Gαi and Gαq to understand the structural basis of the M1 receptor’s preference for the Gαq protein and the M2 receptor’s preference for the Gαi protein. The MD studies showed that the M1 and M2 receptors can couple to both Gα proteins such that the M1 receptor engages with the two Gα proteins in slightly different orientations and the M2 receptor engages with the two Gα proteins in the same orientation. Thermodynamic studies of the free energy of binding of the receptors to the Gα proteins showed that the M1 and M2 receptors bind more strongly to their cognate Gα proteins compared to their non‐cognate ones, which is in line with previous experimental studies on the M3 receptor. A detailed analysis of receptor–G protein interactions showed some cognate‐complex‐specific interactions for the M2:Gαi complex; however, G protein selectivity determinants are spread over a large overlapping subset of residues. Conserved interaction between transmembrane helices 5 and 6 far away from the G‐protein‐binding receptor interface was found only in the two cognate complexes and not in the non‐cognate complexes. An analysis of residues implicated previously in G protein selectivity, in light of the cognate and non‐cognate structures, shaded a more nuanced role of those residues in affecting G protein selectivity. The simulation of both cognate and non‐cognate receptor–G protein complexes fills a structural gap due to difficulties in determining non‐cognate complex structures and provides an enhanced framework to probe the mechanisms of G protein selectivity exhibited by most GPCRs.Support or Funding InformationThis research was funded in part by the California State University Program for Education & Research in Biotechnology (CSUPERB) New Investigator Grant to R.A. Student support for L.J.S. was provided by the NIH Research Initiative for Scientific Enhancement (RISE) grant (GM063787) to CSUN.Strong noncovalent interactions between receptors and Gα proteins mapped to individual domains in the receptor for the complexes: (A) M1:Gαi, (B) M1:Gαq, (C) M2:Gαi, and (D) M2:Gαq. The size of the receptor domain is proportional to the number of its contacts with the Gα protein. The structure of the Gα protein is shown but the receptor is only represented by circles for strongly interacting transmembrane (TM) domains and ovals for non‐TM domains.Figure 1

Antinociceptive effects of potent, selective and brain penetrant muscarinic M4 positive allosteric modulators in rodent pain models

Analgesic properties of orthosteric agonists of the muscarinic M4 receptor subtype have been documented in literature reports, with evidence from pharmacological and in vivo receptor knock out (KO) studies. Constitutive M4 receptor KO mice demonstrated an increased response in the formalin pain model, supporting this hypothesis. Two novel positive allosteric modulators (PAM) of the M4 receptor, Compounds 1 and 2, were characterized in rodent models of acute nociception. Results indicated decreased time spent on nociceptive behaviors in the mouse formalin model, and efficacy in the mouse tail flick assay. The analgesic-like effects of Compounds 1 and 2 were shown to be on target, as the compounds lacked any activity in constitutive M4 KO mice, while retaining activity in wild type control littermates. The analgesic-like effects of Compounds 1 and 2 were significantly diminished in KO mice that have selective deletion of the M4 receptor in neurons that co-express the dopaminergic D1 receptor subtype, suggesting a centrally-mediated effect on nociception. The opioid antagonist naloxone did not diminish the effect of Compound 1, indicating the effects of Compound 1 are not secondarily linked to opioid pathways. Compound 1 was evaluated in the rat, where it demonstrated analgesic-like effects in tail flick and a subpopulation of spinal nociceptive sensitive neurons, suggesting some involvement of spinal mechanisms of nociceptive modulation. These studies indicate that M4 PAMs may be a tractable target for pain management assuming an appropriate safety profile, and it appears likely that both spinal and supraspinal pathways may mediate the antinociceptive-like effects.