Role Of M1 Muscarinic Receptors Research Articles

Mechanisms of Organophosphate Toxicity and the Role of Acetylcholinesterase Inhibition.

Organophosphorus compounds (OPs) have applications in agriculture (e.g., pesticides), industry (e.g., flame retardants), and chemical warfare (nerve agents). In high doses or chronic exposure, they can be toxic or lethal. The primary mechanism, common among all OPs, that initiates their toxic effects is the inhibition of acetylcholinesterase. In acute OP exposure, the subsequent surge of acetylcholine in cholinergic synapses causes a peripheral cholinergic crisis and status epilepticus (SE), either of which can lead to death. If death is averted without effective seizure control, long-term brain damage ensues. This review describes the mechanisms by which elevated acetylcholine can cause respiratory failure and trigger SE; the role of the amygdala in seizure initiation; the role of M1 muscarinic receptors in the early stages of SE; the neurotoxic pathways activated by SE (excitotoxicity/Ca++ overload/oxidative stress, neuroinflammation); and neurotoxic mechanisms linked to low-dose, chronic exposure (Ca++ dyshomeostasis/oxidative stress, inflammation), which do not depend on SE and do not necessarily involve acetylcholinesterase inhibition. The evidence so far indicates that brain damage from acute OP exposure is a direct result of SE, while the neurotoxic mechanisms activated by low-dose chronic exposure are independent of SE and may not be associated with acetylcholinesterase inhibition.

Diverse glutamatergic inputs target spines expressing M1 muscarinic receptors in the basolateral amygdala: An ultrastructural analysis

Although it is known that acetylcholine acting through M1 muscarinic receptors (M1Rs) is essential for memory consolidation in the anterior basolateral nucleus of the amygdala (BLa), virtually nothing is known about the circuits involved. In the hippocampus M1R activation facilitates long-term potentiation (LTP) by potentiating NMDA glutamate receptor (NMDAR) currents. The majority of NMDAR+ profiles in the BLa are spines. Since about half of dendritic spines of BLa pyramidal neurons (PNs) receiving glutamatergic inputs are M1R-immunoreactive (M1R+) it is possible that the role of M1Rs in BLa mnemonic functions also involves potentiation of NMDAR currents in spines. However, the finding that only about half of BLa spines are M1R+ suggests that this proposed mechanism may only apply to a subset of glutamatergic inputs. As a first step in the identification of differential glutamatergic inputs to M1R+ spines in the BLa, the present electron microscopic study used antibodies to two different vesicular glutamate transporter proteins (VGluTs) to label two different subsets of glutamatergic inputs to M1R+ spines. These inputs are largely complimentary with VGluT1+ inputs arising mainly from cortical structures and the basolateral nucleus, and VGluT2+ inputs arising mainly from the thalamus. It was found that about one-half of the spines that were postsynaptic to VGluT1+ or VGluT2+ terminals were M1R+. In addition, a subset of the VGluT1+ or VGluT2+ axon terminals were M1R+, including those that synapsed with M1R+ spines. These results suggest that acetylcholine can modulate glutamatergic inputs to BLa spines by presynaptic as well as postsynaptic M1R-mediated mechanisms.

M1 muscarinic receptors facilitate hippocampus-dependent cognitive flexibility via modulating GluA2 subunit of AMPA receptors

Cognitive flexibility is an important aspect of executive function. The cholinergic system, an important component of cognition, has been shown to modulate cognitive flexibility mainly through the striatum and prefrontal cortex. The role of M1 muscarinic receptors (M1 mAChRs), an important therapeutic target in the cholinergic system, in hippocampus-dependent cognitive flexibility is unclarified. In the present study, we demonstrated that selective activation of M1 mAChRs promoted extinction of initial learned response and facilitated acquisition of reversal learning in the Morris water maze, a behavior test that is mainly dependent on the hippocampus. However, these effects were abolished in GluA2 mutant mice with deficiency in phosphorylation of Ser880 by protein kinase C (PKC). Further long-term depression (LTD) in the hippocampal CA1 area induced by M1 mAChR activation was shown to be dependent on AMPA receptor subunit GluA2 but not GluA1. M1 mAChRs increased GluA2 endocytosis through phosphorylation of Ser880 by PKC. Inhibition of PKC blocked M1 mAChR-mediated LTD, memory switching and reversal learning facilitation. Moreover, the slow memory extinction observed in GluA2 mutant mice and PKC inhibitor-treated mice appeared to affect the consolidation and retrieval of reversal learning. Thus, these results demonstrate that M1 mAChRs mainly facilitate acquisition in spatial reversal learning and further elucidate that such an effect is dependent on the phosphorylation of GluA2 by PKC. The study helps clarify the role of M1 mAChRs in cognitive flexibility and may prompt the earlier prevention of cognitive inflexibility.

Data regarding M1 muscarinic receptor-mediated modulation of hepatic catalase activity in response to oxidative stress.

We recently demonstrated the role of M1 muscarinic receptors (M1R) in modulating oxidative stress in liver and hepatocytes (Urrunaga et al., 2015) [1]. Here we provide data regarding the effect of a novel M1R agonist, VU0357017 (Lebois et al., 2010) [2], on H2O2-mediated hepatocyte injury, the effect of an M1R antagonist VU0255035 (Sheffler et al., 2009) [3] on catalase and super oxide dismutase (SOD) activities in H2O2–treated hepatocytes in vitro, and finally, the effect of M1R ablation on hepatic catalase activity in acetaminophen (APAP)-treated mice.

M1 Muscarinic Receptor Deficiency Attenuates Azoxymethane-Induced Chronic Liver Injury in Mice.

Cholinergic nervous system regulates liver injury. However, the role of M1 muscarinic receptors (M1R) in modulating chronic liver injury is uncertain. To address this gap in knowledge we treated M1R-deficient and WT mice with azoxymethane (AOM) for six weeks and assessed liver injury responses 14 weeks after the last dose of AOM. Compared to AOM-treated WT mice, M1R-deficient mice had attenuated liver nodularity, fibrosis and ductular proliferation, α-SMA staining, and expression of α1 collagen, Tgfβ-R, Pdgf-R, Mmp-2, Timp-1 and Timp-2. In hepatocytes, these findings were associated with reductions of cleaved caspase-3 staining and Tnf-α expression. In response to AOM treatment, M1R-deficient mice mounted a vigorous anti-oxidant response by upregulating Gclc and Nqo1 expression, and attenuating peroxynitrite generation. M1R-deficient mouse livers had increased expression of Trail-R2, a promotor of stellate cell apoptosis; dual staining for TUNNEL and α-SMA revealed increased stellate cells apoptosis in livers from M1R-deficient mice compared to those from WT. Finally, pharmacological inhibition of M1R reduced H2O2-induced hepatocyte apoptosis in vitro. These results indicate that following liver injury, anti-oxidant response in M1R-deficient mice attenuates hepatocyte apoptosis and reduces stellate cell activation, thereby diminishing fibrosis. Therefore, targeting M1R expression and activation in chronic liver injury may provide therapeutic benefit.

Anticholinergic drugs rescue synaptic plasticity in DYT1 dystonia: role of M1 muscarinic receptors.

Broad-spectrum muscarinic receptor antagonists have represented the first available treatment for different movement disorders such as dystonia. However, the specificity of these drugs and their mechanism of action is not entirely clear. We performed a systematic analysis of the effects of anticholinergic drugs on short- and long-term plasticity recorded from striatal medium spiny neurons from DYT1 dystonia knock-in (Tor1a(+/Δgag) ) mice heterozygous for ΔE-torsinA and their controls (Tor1a(+/+) mice). Antagonists were chosen that had previously been proposed to be selective for muscarinic receptor subtypes and included pirenzepine, trihexyphenydil, biperiden, orphenadrine, and a novel selective M1 antagonist, VU0255035. Tor1a(+/Δgag) mice exhibited a significant impairment of corticostriatal synaptic plasticity. Anticholinergics had no significant effects on intrinsic membrane properties and on short-term plasticity of striatal neurons. However, they exhibited a differential ability to restore the corticostriatal plasticity deficits. A complete rescue of both long-term depression (LTD) and synaptic depotentiation (SD) was obtained by applying the M1 -preferring antagonists pirenzepine and trihexyphenidyl as well as VU0255035. Conversely, the nonselective antagonist orphenadrine produced only a partial rescue of synaptic plasticity, whereas biperiden and ethopropazine failed to restore plasticity. The selectivity for M1 receptors was further demonstrated by their ability to counteract the M1 -dependent potentiation of N-methyl-d-aspartate (NMDA) current recorded from striatal neurons. Our study demonstrates that selective M1 muscarinic receptor antagonism offsets synaptic plasticity deficits in the striatum of mice with the DYT1 dystonia mutation, providing a potential mechanistic rationale for the development of improved antimuscarinic therapies for this movement disorder.

Interaction between glutamatergic–NMDA and cholinergic–muscarinic systems in classical fear conditioning

A number of studies have suggested that the glutamatergic and cholinergic systems are both involved in learning and memory processes and that they interact in order to facilitate these processes. However, the role of M1-muscarinic receptors in mediating this interaction has not been elucidated. The aim of this study was to determine whether the concomitant administration of MK-801 (non-competitive NMDA antagonist) and dicyclomine (M1-muscarinic antagonist – DIC) in sub-effective doses impairs contextual fear conditioning (hippocampal-dependent task) and tone fear conditioning tasks (hippocampal-independent task). The results showed that concomitant pre-training administration of DIC (8.0 mg/kg) and MK-801 (0.07 mg/kg) – two sub-effectives doses for the contextual fear conditioning task – does impair the performance of animals on this task (as measured by freezing behavior time). Tone fear conditioning tasks were not affected by the drugs either administered separately or concurrently. The pre-training administration of sub-effective doses of MK-801 and DIC in combination impairs performance on contextual fear conditioning task (hippocampal-dependent), but not on tone fear conditioning task (hippocampal-independent). These data support the hypothesis that the interaction between glutamatergic and cholinergic systems in hippocampus-dependent learning and memory processes probably occurs through M1 receptor.

Blockade of M1 muscarinic acetylcholine receptors modulates the methamphetamine-induced psychomotor stimulant effect

Muscarinic acetylcholine receptors (M1–M5) regulate many key functions of the CNS and peripheral nervous system. In the present study, the role of M1 muscarinic receptors (M1R) in the psychomotor stimulant and sensitizing properties of methamphetamine (METH) is investigated using molecular, neurochemical, and behavioral approaches. Acute and repeated treatment with METH increased M1R mRNA expression in the frontal cortex and the CA2 region of the hippocampus. Repeated treatment with METH also increased M1R mRNA expression in the dentate gyrus. Dicyclomine, an M1R antagonist, did not affect the psychomotor effect of METH, but it attenuated METH-induced increases in the dopamine (DA) efflux in the nucleus accumbens (NAc). Dicyclomine enhanced the psychomotor effect of METH after repeated treatment with METH and 8.0 mg/kg of dicyclomine, and also augmented the increase in the NAc DA overflow evoked by repeated METH treatment. These results suggest that M1R plays a role in the METH-induced psychomotor stimulant effect by changing the release of DA in the NAc of mice.

Muscarinic Receptors Control Postprandial Release of Glucagon-Like Peptide-1: In Vivo and in Vitro Studies in Rats

Plasma levels of glucagon-like peptide-1 (GLP-1) rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve that stimulates the L cell in the distal gut. The role of muscarinic receptors in this pathway was thus investigated using the anesthetized rat and fetal rat intestinal cells (FRIC) in culture. GLP-1 secretion from the distal gut increased 5-fold after 3 ml corn oil were placed into the proximal duodenum (P < 0.001). Atropine (a nonspecific muscarinic receptor antagonist) completely inhibited fat-induced GLP-1 secretion in vivo (P < 0.01). Pirenzepine (an M1 muscarinic receptor antagonist) also inhibited fat-induced GLP-1 secretion in vivo, by 91 +/- 6% (P < 0.01). Gallamine (an M2 muscarinic receptor antagonist) and 4-diphenylacetoxy-N-methylpiperidine (an M3 muscarinic receptor antagonist) had no effect. Incubating FRIC cultures with bethanechol (a muscarinic receptor agonist) stimulated GLP-1 secretion to 200 +/- 22% of control (P < 0.01). Pirenzepine and gallamine significantly inhibited bethanechol-stimulated GLP-1 secretion, by 96 +/- 12% and 98 +/- 8%, respectively (P < 0.01). Unexpectedly, 4-diphenylacetoxy-N-methylpiperidine stimulated GLP-1 secretion by FRIC cells, to 324 +/- 52% of the control value (P < 0.01). Double immunofluorescent staining using GLP-1 and M1, M2, and M3 muscarinic receptor antibodies showed expression of the three subtypes of muscarinic receptors by the L cells in rat ileal sections and FRIC cultures. These results demonstrate the role of M1 muscarinic receptors expressed by L cells in the control of postprandial secretion of GLP-1. M2 muscarinic receptors also seem to play a role in controlling GLP-1 secretion by fetal, but not adult, L cells.

Cholinergic activity modulates the survival of retinal ganglion cells in culture: the role of M1 muscarinic receptors

The control of natural cell death is mediated by neurotrophins released by target, afferent and glial cells. In the present work we show that treatment of retinal cells ‘in vitro’ for 48 h with 25 μM carbamylcholine induced a two-fold increase in retinal ganglion cells survival. This effect was dose-dependent and mediated by M1 receptors since it could be blocked by 1 μM telenzepine (a M1 receptor antagonist) and mimicked by 200 μM oxotremorine (a M1 receptor agonist). The effect of carbamylcholine was abolished by 10 μM BAPTA-AM (an intracellular Ca2+ chelator), 30 μM dantrolene (an inhibitor of ryanodinic receptors), 500 nM H-89 (an inhibitor of PKA), 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 50 μM PD-98059 (a MEK inhibitor). Treatment with 10 μM genistein (an inhibitor of tyrosine kinase), 25 μM LY-294002 (a PI-3 kinase blocker), 30 nM brefeldin-A (a blocker of polypeptides release), 50 nM K-252a (a Trk receptor inhibitor) and 20 μM fluorodeoxyuridine (an inhibitor of cell proliferation) totally inhibited the effect of carbamylcholine. Taken together our results indicate that muscarinic activity controls the survival of retinal ganglion cells through a mechanism involving the release of polypeptides and activation of Irk receptors.

Pirenzepine Blunts the Pulmonary Parenchymal Response to Inhaled Methacholine

Summary: To determine the role of M1 muscarinic receptors in the response of the pulmonary parenchyma to inhaled methacholine (MCh), 20 mongrel, out-bred puppies, 8-10 weeks of age were challenged following pretreatment with either saline (control), UH-AH37 (a combined M1 & M3 receptor blocker), or pirenzepine (a relatively selective M1 receptor blocker). In addition, eight fox hound-beagle puppies, born and raised in a clean animal house, were studied. Relatively selective doses of pirenzepine produced a dose-dependent shift to the right of the parenchymal dose-response curves ( P=0.031), with no effect on the airway dose-response curve ( P=0.102). The fox hound-beagle puppies showed less parenchymal response ( P<0.0005), but equivalent airway response ( P=0.468), to MCh compared with the mongrel puppies. High doses of pirenzepine (10000 μg/kg) and UH-AH37 (3 mg/kg) markedly inhibited both the parenchymal and airway responses to MCh. Data from the present study demonstrate that: (1) while both the airway and pulmonary parenchyma respond to inhaled MCh, the mechanisms by which they respond differ; (2) stimulation of M1 subtype muscarinic receptors are responsible, at least partly, for the parenchymal response; and (3) experimental conditions, such as the breed and housing conditions of animals, may have major influences on the parenchymal response to inhalational challenge tests.

Binding of muscarinic toxins MT×1 and MT×2 from the venom of the green mamba Dendroaspis angusticeps to cloned human muscarinic cholinoceptors

Muscarinic toxins MTx1 and MTx2 are 7500 mol. wt polypeptides isolated from the venom of the green mamba snake Dendroaspis angusticeps. Previous competition binding studies indicate that the MTxs may be selective for the M, subtype of muscarinic acetylcholine receptors. The present work was undertaken in order to clarify the muscarinic subtype specificity and functional effects of MTx1 and MTx2. Binding interactions were determined using 3 H- N-methyl scopolamine (NMS) and cloned human muscarinic receptor subtypes m1, m2, m3 and m4. Some preliminary functional studies were performed on rabbit vas deferens preparations, which contain M 1 cholinoceptors. MTx1 and MTx2 inhibited 3H-NMS binding to m1 and m3 receptors, with little effect on binding to m2 and m4 receptors. Affinity was higher for ml receptors: K for MTx1 were 48 nM at m1 receptors and 72 nM at m3 receptors, and K i for MTx2 were 364 nM at m1 and 1.2μM at m3 receptors. At m1 receptors, about 90% of the binding of MTx1 and MTx2 appears to be irreversible. On rabbit vas deferens preparations, MTx1 and MTx2 at concentrations above 50 nM behaved in a similar way to the relatively selective M 1-agonists M cN-A-343 and CPCP (4-[ N-(4-chlorophenyl)carba-moyloxy]-4-pent-2-ynyl-trimethylammonium iodide) by reducing responses to nerve stimulation. The results confirm that MTx1 and MTx2 bind to m1 receptors rather than to m2 or m4 receptors, but they also reveal a slightly weaker effect at m3 receptors. The interaction at m1 receptors apears to be essentially irreversible, implying that the toxins could be useful tools in studies of the functional role of m1 muscarinic receptors.

Role of M1 muscarinic receptors in the parasympathetic control of colonic motility in cats and rabbits.

1. The effects of pirenzepine (a selective blocking agent of M1 muscarinic receptors) were studied on excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) elicited on colonic smooth muscle by stimulation of efferent parasympathetic nerve fibres in anaesthetized cats and rabbits. 2. Pirenzepine (25 micrograms kg-1 to 0.2 mg kg-1, i.v.) decreased the amplitude of EJPs or abolished them. In pirenzepine, parasympathetic stimulation elicited IJPs in most cases. 3. In both species, pirenzepine initiated spontaneous IJPs, indicating an increase in the activity of the non-adrenergic, non-cholinergic (NANC) inhibitory neurones. 4. The results suggest that M1 muscarinic receptors are involved in synaptic transmission within intramural plexuses, at interneuronal synapses in the parasympathetic excitatory pathway to colonic smooth muscle, but are not involved in the pathway to the NANC inhibitory neurones. The facilitation of IJPs by pirenzepine suggests that, under normal physiological conditions, NANC neurones are tonically inhibited by an intramural nervous circuit involving M1 muscarinic receptors.