mAChR Subtypes Research Articles

Structural Characteristics of the Allosteric Binding Site Represent a Key to Subtype Selective Modulators of Muscarinic Acetylcholine Receptors.

The high conservation of the orthosteric acetylcholine binding site of muscarinic receptors (MAChR) represents a considerable challenge in terms of designing subtype selective drugs. A promising approach to gain subtype selectivity is to include allosteric or dualsteric targeting that aims to address more specific extracellular binding sites. Despite recent advances in crystallography of G protein coupled receptors (GPCRs), structural information for all 5 MAChR subtypes is not yet available. Here we report structural models of the active and the inactive receptor state of all subtypes derived by homology modelling in combination with MD simulations. The comparison of the allosteric binding site unveils the characteristics for each subtype on a structural level and indicates anchor points for rational design of selective drugs. Additionally, homology models offer the possibility for a rational explanation of dualsteric subtype selectivity, as we show for the M2 over M5 selectivity of the dualsteric ligands Atr-6-naph and Iper-6-phth.

Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model

Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M1 positive allosteric modulator (PAM), VU0453595, allowed us to evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia.

The Neurotoxic Effect of 13,19-Didesmethyl and 13-Desmethyl Spirolide C Phycotoxins Is Mainly Mediated by Nicotinic Rather Than Muscarinic Acetylcholine Receptors.

Spirolides are a large family of lipophilic marine toxins produced by dinoflagellates that have been detected in contaminated shellfish. Among them, 13,19-didesmethyl and 13-desmethyl spirolide C phycotoxins are widely distributed and their mode of action needs to be clearly defined. In order to further characterize the pharmacological profiles of these phycotoxins on various nicotinic acetylcholine receptor (nAChR) subtypes and to examine whether they act on muscarinic receptors (mAChRs), functional electrophysiological studies and competition binding experiments have been performed. While 13-desmethyl spirolide C interacted efficiently with sub-nanomolar affinities and low selectivity with muscular and neuronal nAChRs, 13,19-didesmethyl spirolide C was more selective of muscular and homopentameric α7 receptors and recognized only weakly neuronal heteropentameric receptors, especially the α4β2 subtype. Thus, the presence of an additional methyl group on the tetrahydropyran ring significantly modified the pharmacological profile of 13-desmethyl spirolide C by notably increasing its affinity on certain neuronal nAChRs. Structural explanations of this selectivity difference are proposed, based on molecular docking experiments modeling different spirolide-receptor complexes. In addition, the 2 spirolides interacted only with low micromolar affinities with the 5 mAChRs, highlighting that the toxicity of the spirolide C analogs is mainly due to their high inhibition potency on various peripheral and central nAChRs and not to their low ability to interact with mAChR subtypes.

The Repellent DEET Potentiates Carbamate Effects via Insect Muscarinic Receptor Interactions: An Alternative Strategy to Control Insect Vector-Borne Diseases.

Insect vector-borne diseases remain one of the principal causes of human mortality. In addition to conventional measures of insect control, repellents continue to be the mainstay for personal protection. Because of the increasing pyrethroid-resistant mosquito populations, alternative strategies to reconstitute pyrethroid repellency and knock-down effects have been proposed by mixing the repellent DEET (N,N-Diethyl-3-methylbenzamide) with non-pyrethroid insecticide to better control resistant insect vector-borne diseases. By using electrophysiological, biochemichal, in vivo toxicological techniques together with calcium imaging, binding studies and in silico docking, we have shown that DEET, at low concentrations, interacts with high affinity with insect M1/M3 mAChR allosteric site potentiating agonist effects on mAChRs coupled to phospholipase C second messenger pathway. This increases the anticholinesterase activity of the carbamate propoxur through calcium-dependent regulation of acetylcholinesterase. At high concentrations, DEET interacts with low affinity on distinct M1/M3 mAChR site, counteracting the potentiation. Similar dose-dependent dual effects of DEET have also been observed at synaptic mAChR level. Additionally, binding and in silico docking studies performed on human M1 and M3 mAChR subtypes indicate that DEET only displays a low affinity antagonist profile on these M1/M3 mAChRs. These results reveal a selective high affinity positive allosteric site for DEET in insect mAChRs. Finally, bioassays conducted on Aedes aegypti confirm the synergistic interaction between DEET and propoxur observed in vitro, resulting in a higher mortality of mosquitoes. Our findings reveal an unusual allosterically potentiating action of the repellent DEET, which involves a selective site in insect. These results open exciting research areas in public health particularly in the control of the pyrethroid-resistant insect-vector borne diseases. Mixing low doses of DEET and a non-pyrethroid insecticide will lead to improvement in the efficiency treatments thus reducing both the concentration of active ingredients and side effects for non-target organisms. The discovery of this insect specific site may pave the way for the development of new strategies essential in the management of chemical use against resistant mosquitoes.

Molecular mechanisms of synergy of corneal muscarinic and nicotinic acetylcholine receptors in upregulation of E-cadherin expression

Corneal epithelial erosion is one of the most common problems in clinical ophthalmology. Despite significant progress in understanding how the cornea heals, clinically available pharmacological therapies that can promote repair and prevent visual complications remain limited. We have recently demonstrated that the acetylcholine (ACh) axis of corneal epithelium plays an important role in regulation and coordination of distinct activities of corneal epithelial cells (CEC) mediating re-epithelialization, but mechanisms remained unclear. We hypothesized that the grounds for synergistic effects of corneal ACh receptors lie within the signaling pathways linking different receptors to specific elements of the CEC pro-epithelialization activities. In this study, we sought to elucidate the molecular mechanisms of cooperation of corneal muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) in upregulation of E-cadherin expression. The roles of individual corneal mAChRs and nAChRs subtypes were investigated by in-cell western assay of the ACh-treated CEC, in which different ACh receptor genes were silenced by receptor-specific shRNAs. Functional inactivation of M3, but not M4, mAChR subtype, or α3 or α7, but not α9, nAChR subunit significantly inhibited E-cadherin expression. To gain a mechanistic insight, we blocked the key steps of the downstream signaling pathways. Results demonstrated that cholinergic agonists can upregulate E-cadherin expression by activating M3 mAChR, and α3β2 and α7 nAChRs via the common signaling cascade Ca2+–CaMKII–PKC–Ras–Raf–MEK–ERK. Activation of α7 nAChR can launch the Ras–Raf–MEK–ERK cascade both indirectly, through the Ca2+–CaMKII–PKC step, and directly, perhaps, due to its direct interaction with Ras. Although the biological significance of such redundancy remained to be elucidated, results of the present study point to a new direction to pharmacologically accelerate corneal re-epithelialization, and should have salient clinical implication.

Structural determinants for the interactions between muscarinic toxin 7 and muscarinic acetylcholine receptors.

Muscarinic acetylcholine receptors (mAChRs) have five subtypes and play crucial roles in various physiological functions and pathophysiological processes. Poor subtype specificity of mAChR modulators has been an obstacle to discover new therapeutic agents. Muscarinic toxin 7 (MT7) is a natural peptide toxin with high selectivity for the M1 receptor. With three to five residues substituted, M3, M4, and M5 receptor mutants could bind to MT7 at nanomolar concentration as the M1 receptor. However, the structural mechanisms explaining MT7-mAChRs binding are still largely unknown. In this study, we constructed 10 complex models of MT7 and each mAChR subtype or its mutant, performed molecular dynamics simulations, and calculated the binding energies to investigate the mechanisms. Our results suggested that the structural determinants for the interactions on mAChRs were composed of some critical residues located separately in the extracellular loops of mAChRs, such as Glu4.56, Leu4.60, Glu/Gln4.63, Tyr4.65, Glu/Asp6.67, and Trp7.35. The subtype specificity of MT7 was attributed to the non-conserved residues at positions 4.56 and 6.67. These structural mechanisms could facilitate the discovery of novel mAChR modulators with high subtype specificity and enhance the understanding of the interactions between ligands and G-protein-coupled receptors.

123I-iododexetimide preferentially binds to the muscarinic receptor subtype M1 in vivo.

The muscarinic M1 receptor (M1R) is highly involved in cognition, and selective M1 agonists have procognitive properties. Loss of M1R has been found in postmortem brain tissue for several neuropsychiatric disorders and may be related to symptoms of cognitive dysfunction. (123)I-iododexetimide is used for imaging muscarinic acetylcholine receptors (mAchRs). Considering its high brain uptake and intense binding in M1R-rich brain areas, (123)I-iododexetimide may be an attractive radiopharmaceutical to image M1R. To date, the binding affinity and selectivity of (123)I-iododexetimide for the mAchR subtypes has not been characterized, nor has its brain distribution been studied intensively. Therefore, this study aimed to address these topics. The in vitro affinity and selectivity of (127)I-iododexetimide (cold-labeled iododexetimide), as well as its functional antagonist properties (guanosine 5'-[γ-(35)S-thio]triphosphate [GTPγ(35)S] assay), were assessed on recombinant human M1R-M5R. Distributions of (127)I-iododexetimide and (123)I-iododexetimide in the brain were evaluated using liquid chromatography-mass spectrometry and storage phosphor imaging, respectively, ex vivo in rats, wild-type mice, and M1-M5 knock-out (KO) mice. Inhibition of (127)I-iododexetimide and (123)I-iododexetimide binding in M1R-rich brain areas by the M1R/M4R agonist xanomeline, or the antipsychotics olanzapine (M1R antagonist) and haloperidol (low M1R affinity), was assessed in rats ex vivo. In vitro, (127)I-iododexetimide displayed high affinity for M1R (pM range), with modest selectivity over other mAchRs. In biodistribution studies on rats, ex vivo (127)I-iododexetimide binding was much higher in M1R-rich brain areas, such as the cortex and striatum, than in cerebellum (devoid of M1Rs). In M1 KO mice, but not M2-M5 KO mice, (127)I-iododexetimide binding was strongly reduced in the frontal cortex compared with wild-type mice. Finally, acute administration of both an M1R/M4R agonist xanomeline and the M1R antagonist olanzapine was able to inhibit (123)I-iododexetimide ex vivo, and (123)I-iododexetimide binding in M1-rich brain areas in rats, whereas administration of haloperidol had no effect. The current results suggest that (123)I-iododexetimide preferentially binds to M1R in vivo and can be displaced by M1R ligands. (123)I-iododexetimide may therefore be a useful imaging tool as a way to further evaluate M1R changes in neuropsychiatric disorders, as a potential stratifying biomarker, or as a clinical target engagement biomarker to assess M1R.

Development of a highly potent, novel M5 positive allosteric modulator (PAM) demonstrating CNS exposure: 1-((1H-indazol-5-yl)sulfoneyl)-N-ethyl-N-(2-(trifluoromethyl)benzyl)piperidine-4-carboxamide (ML380).

A functional high throughput screen identified a novel chemotype for the positive allosteric modulation (PAM) of the muscarinic acetylcholine receptor (mAChR) subtype 5 (M5). Application of rapid analog, iterative parallel synthesis efficiently optimized M5 potency to arrive at the most potent M5 PAMs prepared to date and provided tool compound 8n (ML380) demonstrating modest CNS penetration (human M5 EC50 = 190 nM, rat M5 EC50 = 610 nM, brain to plasma ratio (Kp) of 0.36).

Selective activation of M4 muscarinic acetylcholine receptors reverses MK-801-induced behavioral impairments and enhances associative learning in rodents.

Positive allosteric modulators (PAMs) of the M4 muscarinic acetylcholine receptor (mAChR) represent a novel approach for the treatment of psychotic symptoms associated with schizophrenia and other neuropsychiatric disorders. We recently reported that the selective M4 PAM VU0152100 produced an antipsychotic drug-like profile in rodents after amphetamine challenge. Previous studies suggest that enhanced cholinergic activity may also improve cognitive function and reverse deficits observed with reduced signaling through the N-methyl-d-aspartate subtype of the glutamate receptor (NMDAR) in the central nervous system. Prior to this study, the M1 mAChR subtype was viewed as the primary candidate for these actions relative to the other mAChR subtypes. Here we describe the discovery of a novel M4 PAM, VU0467154, with enhanced in vitro potency and improved pharmacokinetic properties relative to other M4 PAMs, enabling a more extensive characterization of M4 actions in rodent models. We used VU0467154 to test the hypothesis that selective potentiation of M4 receptor signaling could ameliorate the behavioral, cognitive, and neurochemical impairments induced by the noncompetitive NMDAR antagonist MK-801. VU0467154 produced a robust dose-dependent reversal of MK-801-induced hyperlocomotion and deficits in preclinical models of associative learning and memory functions, including the touchscreen pairwise visual discrimination task in wild-type mice, but failed to reverse these stimulant-induced deficits in M4 KO mice. VU0467154 also enhanced the acquisition of both contextual and cue-mediated fear conditioning when administered alone in wild-type mice. These novel findings suggest that M4 PAMs may provide a strategy for addressing the more complex affective and cognitive disruptions associated with schizophrenia and other neuropsychiatric disorders.

Mode of interaction of 1,4-dioxane agonists at the M2 and M3 muscarinic receptor orthosteric sites

The methyl group in cis stereochemical relationship with the basic chain of all pentatomic cyclic analogues of ACh is crucial for the agonist activity at mAChR. Among these only cevimeline (1) is employed in the treatment of xerostomia associated with Sjögren’s syndrome. Here we demonstrated that, unlike 1,3-dioxolane derivatives, in the 1,4-dioxane series the methyl group is not essential for the activation of mAChR subtypes. Docking studies, using the crystal structures of human M2 and rat M3 receptors, demonstrated that the 5-methylene group of the 1,4-dioxane nucleus of compound 10 occupies the same lipophilic pocket as the methyl group of the 1,3-dioxolane 4.

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.

M1 muscarinic acetylcholine receptor in Alzheimer’s disease

The degeneration of cholinergic neurons and cholinergic hypofunction are pathologies associated with Alzheimer's disease (AD). Muscarinic acetylcholine receptors (mAChRs) mediate acetylcholine-induced neurotransmission and five mAChR subtypes (M1-M5) have been identified. Among them, M1 mAChR is widely expressed in the central nervous system and has been implicated in many physiological and pathological brain functions. In addition, M1 mAChR is postulated to be an important therapeutic target for AD and several other neurodegenerative diseases. In this article, we review recent progress in understanding the functional involvement of M1 mAChR in AD pathology and in developing M1 mAChR agonists for AD treatment.

M4 mAChR-Mediated Modulation of Glutamatergic Transmission at Corticostriatal Synapses

The striatum is the main input station of the basal ganglia and is extensively involved in the modulation of motivated behavior. The information conveyed to this subcortical structure through glutamatergic projections from the cerebral cortex and thalamus is processed by the activity of several striatal neuromodulatory systems including the cholinergic system. Acetylcholine potently modulates glutamate signaling in the striatum via activation of muscarinic receptors (mAChRs). It is, however, unclear which mAChR subtype is responsible for this modulatory effect. Here, by using electrophysiological, optogenetic, and immunoelectron microscopic approaches in conjunction with a novel, highly selective M4 positive allosteric modulator VU0152100 (ML108) and M4 knockout mice, we show that M4 is a major mAChR subtype mediating the cholinergic inhibition of corticostriatal glutamatergic input on both striatonigral and striatopallidal medium spiny neurons (MSNs). This effect is due to activation of presynaptic M4 receptors, which, in turn, leads to a decrease in glutamate release from corticostriatal terminals. The findings of the present study raise the interesting possibility that M4 mAChR could be a novel therapeutic target for the treatment of neurological and neuropsychiatric disorders involving hyper-glutamatergic transmission at corticostriatal synapses.

Evaluation of 1,2,5-thiadiazoles as modulators of M1/M5 muscarinic receptor subtypes

Studies have demonstrated the presence of allosteric binding sites on each of the muscarinic acetylcholine receptor (mAChR) subtypes. Since most drugs targeting muscarinic receptors bind to the highly conserved orthosteric binding site, they fail to achieve appreciable subtype selectivity. Targeting non-conserved allosteric sites may provide a new way of enhancing selectivity for individual subtypes of muscarinic receptor. Tetra(ethyleneglycol)(3-methoxy-1,2,5-thiadiazol-4-yl)[3-(1-methyl-1,2,5,6-tetrahydropyrid-3-yl)-1,2,5-thiadiazol-4-yl] ether, CDD-0304 (10), was found to be a M1/2/4 selective muscarinic agonist and might prove useful in treating the symptoms associated with schizophrenia (J. Med. Chem.2003, 46, 4273). It was hypothesized that the observed subtype selectivity demonstrated by 10 may be due to its ability to function as a bitopic ligand (J. Med. Chem.2006, 49, 7518). To further investigate this possibility, a novel series of compounds was synthesized using a 1,2,5-thiadiazole moiety along with varying lengths of a polyethylene glycol linker and terminal groups, for evaluation as potential allosteric modulators of muscarinic receptors. Preliminary biological studies were performed using carbachol to stimulate M1 and M5 receptors. No significant agonist activity was observed at either M1 or M5 receptors for any of the compounds. Compound 18, 2-(4-methoxy-1,2,5-thiadiazol-3-yloxy)-N,N-dimethylethanamine fumarate (CDD-0361F) was found to block the effects of carbachol at M5 muscarinic receptors.

Antipsychotic Drug-Like Effects of the Selective M4 Muscarinic Acetylcholine Receptor Positive Allosteric Modulator VU0152100

Accumulating evidence suggests that selective M4 muscarinic acetylcholine receptor (mAChR) activators may offer a novel strategy for the treatment of psychosis. However, previous efforts to develop selective M4 activators were unsuccessful because of the lack of M4 mAChR subtype specificity and off-target muscarinic adverse effects. We recently developed VU0152100, a highly selective M4 positive allosteric modulator (PAM) that exerts central effects after systemic administration. We now report that VU0152100 dose-dependently reverses amphetamine-induced hyperlocomotion in rats and wild-type mice, but not in M4 KO mice. VU0152100 also blocks amphetamine-induced disruption of the acquisition of contextual fear conditioning and prepulse inhibition of the acoustic startle reflex. These effects were observed at doses that do not produce catalepsy or peripheral adverse effects associated with non-selective mAChR agonists. To further understand the effects of selective potentiation of M4 on region-specific brain activation, VU0152100 alone and in combination with amphetamine were evaluated using pharmacologic magnetic resonance imaging (phMRI). Key neural substrates of M4-mediated modulation of the amphetamine response included the nucleus accumbens (NAS), caudate-putamen (CP), hippocampus, and medial thalamus. Functional connectivity analysis of phMRI data, specifically assessing correlations in activation between regions, revealed several brain networks involved in the M4 modulation of amphetamine-induced brain activation, including the NAS and retrosplenial cortex with motor cortex, hippocampus, and medial thalamus. Using in vivo microdialysis, we found that VU0152100 reversed amphetamine-induced increases in extracellular dopamine levels in NAS and CP. The present data are consistent with an antipsychotic drug-like profile of activity for VU0152100. Taken together, these data support the development of selective M4 PAMs as a new approach to the treatment of psychosis and cognitive impairments associated with psychiatric disorders such as schizophrenia.

Activation of M1 and M4 muscarinic receptors as potential treatments for Alzheimer's disease and schizophrenia

Alzheimer’s disease (AD) and schizophrenia (SZ) are neurological disorders with overlapping symptomatology, including both cognitive deficits and behavioral disturbances. Current clinical treatments for both disorders have limited efficacy accompanied by dose-limiting side effects, and ultimately fail to adequately address the broad range of symptoms observed. Novel therapeutic options for AD and SZ are needed to better manage the spectrum of symptoms with reduced adverse-effect liability. Substantial evidence suggests that activation of muscarinic acetylcholine receptors (mAChRs) has the potential to treat both cognitive and psychosis-related symptoms associated with numerous central nervous system (CNS) disorders. However, use of nonselective modulators of mAChRs is hampered by dose-limiting peripheral side effects that limit their clinical utility. In order to maintain the clinical efficacy without the adverse-effect liability, efforts have been focused on the discovery of compounds that selectively modulate the centrally located M1 and M4 mAChR subtypes. Previous drug discovery attempts have been thwarted by the highly conserved nature of the acetylcholine site across mAChR subtypes. However, current efforts by our laboratory and others have now focused on modulators that bind to allosteric sites on mAChRs, allowing these compounds to display unprecedented subtype selectivity. Over the past couple of decades, the discovery of small molecules capable of selectively targeting the M1 or M4 mAChR subtypes has allowed researchers to elucidate the roles of these receptors in regulating cognitive and behavioral disturbances in preclinical animal models. Here, we provide an overview of these promising preclinical and clinical studies, which suggest that M1- and M4-selective modulators represent viable novel targets with the potential to successfully address a broad range of symptoms observed in patients with AD and SZ.

Insulin Oversecretion in MSG-Obese Rats is Related to Alterations in Cholinergic Muscarinic Receptor Subtypes in Pancreatic Islets

Background/ Aims: Impaired pancreatic beta cell function and insulin secretion/action are a link between obesity and type 2 diabetes, which are worldwide public health burdens. We aimed to characterize the muscarinic acetylcholine receptor (mAChR) M₁-M₄ subtypes in isolated pancreatic islets from pre-diabetic obese rats that had been treated neonatally with monosodium L-glutamate (MSG). Methods: At 90 days of age, both the MSG and the control groups underwent biometric and biochemical evaluation. Anti-muscarinic drugs were used to study mAChR function either in vivo or in vitro. Results: The results demonstrated that atropine treatment reduced insulin secretion in the MSG-treated and control groups, whereas treatment with an M₂mAChR-selective antagonist increased secretion. Moreover, the insulinostatic effect of an M₃mAChR-selective antagonist was significantly higher in the MSG-treated group. M₁mAChR and M₃mAChR expression was increased in the MSG-obese group by 55% and 73%, respectively. In contrast, M₂mAChR expression decreased by 25% in the MSG group, whereas M₄mAChR expression was unchanged. Conclusions: Functional changes in and altered content of the mAChR (M₁-M₄) subtypes are pivotal to the demand for high pancreatic beta cell insulin secretion in MSG-obese rats, which is directly associated with vagal hyperactivity and peripheral insulin resistance.

Functional Activation of G-Proteins Coupled With Muscarinic Acetylcholine Receptors in Rat Brain Membranes

The functional activation of Gi/o proteins coupled to muscarinic acetylcholine receptors (mAChRs) was investigated with the conventional guanosine-5'-O-(3-[(35)S]thio) triphosphate ([(35)S]GTPγS) binding assay in rat brain membranes. The most efficacious stimulation elicited by acetylcholine or carbachol (CCh) was obtained in striatal membranes. The pharmacological properties of mAChR-mediated [(35)S]GTPγS binding determined with a series of muscarinic agonists and antagonists were almost identical among the three brain regions investigated, i.e., cerebral cortex, hippocampus, and striatum, except for the apparent partial agonist effects of (αR)-α-cyclopentyl-α-hydroxy-N-[1-(4-methyl-3-pentenyl)-4-piperidinyl]benzeneacetamide fumarate (J 104129) observed only in the hippocampus, but not in the other two regions. Among the muscarinic toxins investigated, only MT3 attenuated CCh-stimulated [(35)S] GTPγS binding. The highly selective allosteric potentiator at the M4 mAChR subtype, 3-amino-N-[(4-chlorophenyl)methyl]-4,6-dimethylthieno[2,3-b]pyridine-2-carboxamide (VU 10010), shifted the concentration-response curve for CCh leftwards as well as upwards. On the other hand, neither thiochrome nor brucine N-oxide was effective. The increases induced by CCh and 5-HT were essentially additive, though not completely, indicating that the mAChRs and 5-HT1A receptors were coupled independently to distinct pools of Gi/o proteins. Collectively, all of the data suggest that functional activation of Gi/o proteins coupled to mAChRs, especially the M4 subtype, is detectable by means of CCh-stimulated [(35)S]GTPγS binding assay in rat discrete brain regions.

The Muscarinic Acetylcholine Receptor Agonist BuTAC Mediates Antipsychotic-Like Effects via the M4 Subtype

The generation of muscarinic acetylcholine receptor (mAChR) subtype-selective compounds has been challenging, requiring use of nonpharmacological approaches, such as genetically engineered animals, to deepen our understanding of the potential that members of the muscarinic receptor subtype family hold as therapeutic drug targets. The muscarinic receptor agonist 'BuTAC' was previously shown to exhibit efficacy in animal models of psychosis, although the particular receptor subtype(s) responsible for such activity was unclear. Here, we evaluate the in vitro functional agonist and antagonist activity of BuTAC using an assay that provides a direct measure of G protein activation. In addition, we employ the conditioned avoidance response paradigm, an in vivo model predictive of antipsychotic activity, and mouse genetic deletion models to investigate which presynaptic mAChR subtype mediates the antipsychotic-like effects of BuTAC. Our results show that, in vitro, BuTAC acts as a full agonist at the M2AChR and a partial agonist at the M1 and M4 receptors, with full antagonist activity at M3- and M5AChRs. In the mouse conditioned avoidance response (CAR) assay, BuTAC exhibits an atypical antipsychotic-like profile by selectively decreasing avoidance responses at doses that do not induce escape failures. CAR results using M2(-/-), M4(-/-), and M2/M4 (M2/M4(-/-)) mice found that the effects of BuTAC were near completely lost in M2/M4(-/-) double-knockout mice and potency of BuTAC was right-shifted in M4(-/-) as compared with wild-type and M2(-/-) mice. The M2/M4(-/-) mice showed no altered sensitivity to the antipsychotic effects of either haloperidol or clozapine, suggesting that these compounds mediate their actions in CAR via a non-mAChR-mediated mechanism. These data support a role for the M4AChR subtype in mediating the antipsychotic-like activity of BuTAC and implicate M4AChR agonism as a potential novel therapeutic mechanism for ameliorating symptoms associated with schizophrenia.