The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

Long-Term-But Not Short-Term-Plasticity at the Mossy Fiber-CA3 Pyramidal Cell Synapse in Hippocampus Is Altered in M1/M3 Muscarinic Acetylcholine Receptor Double Knockout Mice.

Muscarinic acetylcholine receptors are well-known for their crucial involvement in hippocampus-dependent learning and memory, but the exact roles of the various receptor subtypes (M1-M5) are still not fully understood. Here, we studied how M1 and M3 receptors affect plasticity at the mossy fiber (MF)-CA3 pyramidal cell synapse. In hippocampal slices from M1/M3 receptor double knockout (M1/M3-dKO) mice, the signature short-term plasticity of the MF-CA3 synapse was not significantly affected. However, the rather unique NMDA receptor-independent and presynaptic form of long-term potentiation (LTP) of this synapse was much larger in M1/M3-deficient slices compared to wild-type slices in both field potential and whole-cell recordings. Consistent with its presynaptic origin, induction of MF-LTP strongly enhanced the excitatory drive onto single CA3 pyramidal cells, with the effect being more pronounced in M1/M3-dKO cells. In an earlier study, we found that the deletion of M2 receptors in mice disinhibits MF-LTP in a similar fashion, suggesting that endogenous acetylcholine employs both M1/M3 and M2 receptors to constrain MF-LTP. Importantly, such synergism was not observed for MF long-term depression (LTD). Low-frequency stimulation, which reliably induced LTD of MF synapses in control slices, failed to do so in M1/M3-dKO slices and gave rise to LTP instead. In striking contrast, loss of M2 receptors augmented LTD when compared to control slices. Taken together, our data demonstrate convergence of M1/M3 and M2 receptors on MF-LTP, but functional divergence on MF-LTD, with the net effect resulting in a well-balanced bidirectional plasticity of the MF-CA3 pyramidal cell synapse.

Role of Conserved Tyrosine Lid Residues in the Activation of the M2 Muscarinic Acetylcholine Receptor.

The development of subtype selective small molecule drugs for the muscarinic acetylcholine receptor (mAChR) family has been challenging. The design of more selective ligands can be improved by understanding the structure and function of key amino acid residues that line ligand binding sites. Here we study the role of three conserved key tyrosine residues [Y1043.33, Y4036.51, and Y4267.39 (Ballesteros and Weinstein numbers in superscript)] at the human M2 mAChR, located at the interface between the orthosteric and allosteric binding sites of the receptor. We specifically focused on the role of the three tyrosine hydroxyl groups in the transition between the inactive and active conformations of the receptor by making phenylalanine point mutants. Single-point mutation at either of the three positions was sufficient to reduce the affinity of agonists by ∼100-fold for the M2 mAChR, whereas the affinity of antagonists remained largely unaffected. In contrast, neither of the mutations affected the efficacy of orthosteric agonists. When mutations were combined into double and triple M2 mAChR mutants, the affinity of antagonists was reduced by more than 100-fold compared with the wild-type M2 receptor. In contrast, the affinity of allosteric modulators, either negative or positive, was retained at all single and multiple mutations, but the degree of allosteric effect exerted on the endogenous ligand acetylcholine was affected at all mutants containing Y4267.39F. These findings will provide insights to consider when designing future mAChR ligands. SIGNIFICANCE STATEMENT: Structural studies demonstrated that three tyrosine residues between the orthosteric and allosteric sites of the M2 muscarinic acetylcholine receptor (mAChR) had different hydrogen bonding networks in the inactive and active conformations. The role of hydroxyl groups of the tyrosine residues on orthosteric and allosteric ligand pharmacology was unknown. We found that hydroxyl groups of the tyrosine residues differentially affected the molecular pharmacology of orthosteric and allosteric ligands. These results provide insights to consider when designing future mAChR ligands.

Associations of common genetic risk variants of the muscarinic acetylcholine receptor M2 with cardiac autonomic dysfunction in patients with schizophrenia.

OBJECTIVES: Decreased vagal modulation, which has consistently been observed in schizophrenic patients, might contribute to increased cardiac mortality in schizophrenia. Previously, associations between CHRM2 (Cholinergic Receptor Muscarinic 2) and cardiac autonomic features have been reported. Here, we tested for possible associations between these polymorphisms and heart rate variability in patients with schizophrenia. METHODS: A total of three single nucleotide polymorphisms (SNPs) in CHRM2 (rs73158705 A>G, rs8191992 T>A and rs2350782 T>C) that achieved significance (p < 5 * 10-8) in genome-wide association studies for cardiac autonomic features were genotyped in 88 drug-naïve patients, 61 patients receiving antipsychotic medication and 144 healthy controls. Genotypes were analysed for associations with parameters of heart rate variability and complexity, in each diagnostic group. RESULTS: We observed a significantly altered heart rate variability in unmedicated patients with identified genetic risk status in rs73158705 A>G, rs8191992 T>A and rs2350782 T>C as compared to genotype non-risk status. In patients receiving antipsychotic medication and healthy controls, these associations were not observed. DISCUSSION: We report novel candidate genetic associations with cardiac autonomic dysfunction in schizophrenia, but larger cohorts are required for replication.

Determination of Region-Specific Roles of the M3 Muscarinic Acetylcholine Receptor in Gastrointestinal Motility.

BACKGROUND: The specific role of the M3 muscarinic acetylcholine receptor in gastrointestinal motility under physiological conditions is unclear, due to a lack of subtype-selective compounds. AIMS: The objective of this study was to determine the region-specific role of the M3 receptor in gastrointestinal motility. METHODS: We developed a novel positive allosteric modulator (PAM) for the M3 receptor, PAM-369. The effects of PAM-369 on the carbachol-induced contractile response of porcine esophageal smooth muscle and mouse colonic smooth muscle (ex vivo) and on the transit in mouse small intestine and rat colon (in vivo) were examined. RESULTS: PAM-369 selectively potentiated the M3 receptor under the stimulation of its orthosteric ligands without agonistic or antagonistic activity. Half-maximal effective concentrations of PAM activity for human, mouse, and rat M3 receptors were 0.253, 0.345, and 0.127 µM, respectively. PAM-369 enhanced carbachol-induced contraction in porcine esophageal smooth muscle and mouse colonic smooth muscle without causing any contractile responses by itself. The oral administration of 30 mg/kg PAM-369 increased the small intestinal transit in both normal motility and loperamide-induced intestinal dysmotility mice but had no effects on the colonic transit, although the M3 receptor mRNA expression is higher in the colon than in the small intestine. CONCLUSIONS: This study provided the first direct evidence that the M3 receptor has different region-specific roles in the motility function between the small intestine and colon in physiological and pathophysiological contexts. Selective PAMs designed for targeted subtypes of muscarinic receptors are useful for elucidating the subtype-specific function.

M2 Muscarinic Receptor-Dependent Contractions of Airway Smooth Muscle are Inhibited by Activation of ß-Adrenoceptors.

Beta-adrenoceptor (ß-AR) agonists inhibit cholinergic contractions of airway smooth muscle (ASM), but the underlying mechanisms are unclear. ASM cells express M3 and M2 muscarinic receptors, but the bronchoconstrictor effects of acetylcholine are believed to result from activation of M3Rs, while the role of the M2Rs is confined to offsetting ß-AR-dependent relaxations. However, a profound M2R-mediated hypersensitization of M3R-dependent contractions of ASM was recently reported, indicating an important role for M2Rs in cholinergic contractions of ASM. Here, we investigated if M2R-dependent contractions of murine bronchial rings were inhibited by activation of ß-ARs. M2R-dependent contractions were apparent at low frequency (2Hz) electric field stimulation (EFS) and short (10s) stimulus intervals. The ß1-AR agonist, denopamine inhibited EFS-evoked contractions of ASM induced by reduction in stimulus interval from 100 to 10 s and was more effective at inhibiting contractions evoked by EFS at 2 than 20 Hz. Denopamine also abolished carbachol-evoked contractions that were resistant to the M3R antagonist 4-DAMP, similar to the effects of the M2R antagonists, methoctramine and AFDX-116. The inhibitory effects of denopamine on EFS-evoked contractions of ASM were smaller in preparations taken from M2R -/- mice, compared to wild-type (WT) controls. In contrast, inhibitory effects of the ß3-AR agonist, BRL37344, on EFS-evoked contractions of detrusor strips taken from M2R -/- mice were greater than WT controls. These data suggest that M2R-dependent contractions of ASM were inhibited by activation of ß1-ARs and that genetic ablation of M2Rs decreased the efficacy of ß-AR agonists on cholinergic contractions.

The Combined Treatment with Chemotherapeutic Agents and the Dualsteric Muscarinic Agonist Iper-8-Naphthalimide Affects Drug Resistance in Glioblastoma Stem Cells.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by heterogeneous cell populations. Among these, the Glioblastoma Stem Cells (GSCs) fraction shares some similarities with Neural Stem Cells. GSCs exhibit enhanced resistance to conventional chemotherapy drugs. Our previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptors (mAChRs) negatively modulates GSCs proliferation and survival. The aim of the present study was to analyze the ability of the M2 dualsteric agonist Iper-8-naphthalimide (N-8-Iper) to counteract GSCs drug resistance. METHODS: Chemosensitivity to M2 dualsteric agonist N-8-Iper and chemotherapy drugs such as temozolomide, doxorubicin, or cisplatin was evaluated in vitro by MTT assay in two different GSC lines. Drug efflux pumps expression was evaluated by RT-PCR and qRT-PCR. RESULTS: By using sub-toxic concentrations of N-8-Iper combined with the individual chemotherapeutic agents, we found that only low doses of the M2 agonist combined with doxorubicin or cisplatin or temozolomide were significantly able to counteract cell growth in both GSC lines. Moreover, we evaluated as the exposure to high and low doses of N-8-Iper downregulated the ATP-binding cassette (ABC) drug efflux pumps expression levels. CONCLUSIONS: Our results revealed the ability of the investigated M2 agonist to counteract drug resistance in two GSC lines, at least partially by downregulating the ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSC-drug resistance in the GBM therapy.

Pioglitazone prevents obesity-related airway hyperreactivity and neuronal M2 receptor dysfunction.

Obesity-related asthma often presents with more severe symptoms than non-obesity-related asthma and responds poorly to current treatments. Both insulin resistance and hyperinsulinemia are common in obesity. We have shown that increased insulin mediates airway hyperreactivity in diet-induced obese rats by causing neuronal M2 muscarinic receptor dysfunction, which normally inhibits acetylcholine release from parasympathetic nerves. Decreasing insulin with streptozotocin prevented airway hyperreactivity and M2 receptor dysfunction. The objective of the present study was to investigate whether pioglitazone, a hypoglycemic drug, prevents airway hyperreactivity and M2 receptor dysfunction in obese rats. Male rats fed a low- or high-fat diet were treated with pioglitazone or PBS by daily gavage. Body weight, body fat, fasting insulin, and bronchoconstriction and bradycardia in response to electrical stimulation of vagus nerves and to aerosolized methacholine were recorded. Pilocarpine, a muscarinic receptor agonist, was used to measure M2 receptor function. Rats on a high-fat diet had potentiated airway responsiveness to vagal stimulation and dysfunctional neuronal M2 receptors, whereas airway responsiveness to methacholine was unaffected. Pioglitazone reduced fasting insulin and prevented airway hyperresponsiveness and M2 receptor dysfunction but did not change inflammatory cytokine mRNA expression in alveolar macrophages. High-fat diet, with and without pioglitazone, had tissue-specific effects on insulin receptor mRNA expression. In conclusion, pioglitazone prevents vagally mediated airway hyperreactivity and protects neuronal M2 muscarinic receptor function in obese rats.

Allostery of atypical modulators at oligomeric G protein-coupled receptors.

Many G protein-coupled receptors (GPCRs) are therapeutic targets, with most drugs acting at the orthosteric site. Some GPCRs also possess allosteric sites, which have become a focus of drug discovery. In the M2 muscarinic receptor, allosteric modulators regulate the binding and functional effects of orthosteric ligands through a mix of conformational changes, steric hindrance and electrostatic repulsion transmitted within and between the constituent protomers of an oligomer. Tacrine has been called an atypical modulator because it exhibits positive cooperativity, as revealed by Hill coefficients greater than 1 in its negative allosteric effect on binding and response. Radioligand binding and molecular dynamics simulations were used to probe the mechanism of that modulation in monomers and oligomers of wild-type and mutant M2 receptors. Tacrine is not atypical at monomers, which indicates that its atypical effects are a property of the receptor in its oligomeric state. These results illustrate that oligomerization of the M2 receptor has functional consequences.

Functions of Muscarinic Receptor Subtypes in Gastrointestinal Smooth Muscle: A Review of Studies with Receptor-Knockout Mice.

Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M2 and M3 subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca2+ mobilisation, myofilament Ca2+ sensitisation, generation of non-selective cationic and chloride currents, K+ current modulation, inhibition or potentiation of voltage-dependent Ca2+ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.

The Combination of the M2 Muscarinic Receptor Agonist and Chemotherapy Affects Drug Resistance in Neuroblastoma Cells.

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.

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.

Silencing of miR490-3p by H. pylori activates DARPP-32 and induces resistance to gefitinib.

Infection with Helicobacter pylori (H. pylori) is the main risk factor for gastric carcinogenesis. In this study, we investigated the expression, molecular functions, and downstream effectors of miR490-3p in gastric cancer. We used in vitro and in vivo models to investigate the role of H. pylori in regulating miR490-3p, DARPP-32-dependent functions, and therapeutic resistance. Human and mouse neoplastic gastric lesions demonstrated a negative correlation between DARPP-32 and miR490-3p expression (R = -0.58, P < 0.01). This was also detected following infection with H. pylori (R = -0.66, P < 0.01). Molecular assays confirmed DARPP-32 as a direct target of miR490-3p. CHRM2, the host gene of miR490-3p, was hypermethylated and downregulated in neoplastic gastric tissues (P < 0.05). H. pylori induced methylation and downregulation of CHRM2 and miR490-3p. Functionally, the reconstitution of miR490-3p sensitized cancer cells to gefitinib by inactivating DRAPP-32-dependent AKT and STAT3 pathways. Patients with low miR490-3p or high DARPP-32 expression had decreased overall survival (P < 0.05). Hypermethylation-mediated silencing of CHRM2 and miR490-3p by H. pylori increased DARPP-32 expression. Downregulation of miR490-3p in gastric cancer plays a role in gefitinib response by inducing DARPP-32-mediated activation of PI3K/AKT, STAT3 signaling pathways.

Identification of Target Genes of Antiarrhythmic Traditional Chinese Medicine Wenxin Keli.

Wenxin Keli (WXKL) is a traditional Chinese medicine drug approved for the treatment of cardiovascular diseases. This study aimed to identify WXKL-targeting genes involved in antiarrhythmic efficacy of WXKL. The Traditional Chinese Medicine Systems Pharmacology (TCMSP) technology platform was used to screen active compounds of WXKL and WXKL-targeting arrhythmia-related genes. A pig model of myocardial ischemia (MI) was established by balloon-expanding the endothelium of the left coronary artery. Pigs were divided into the model group and WXKL group (n = 6). MI, QT interval, heart rate, and arrhythmia were recorded, and the mRNA expression of target genes in myocardial tissues was detected by PCR. Eleven active ingredients of WXKL and eight WXKL-targeting arrhythmia-related genes were screened. Five pathways were enriched, and an "ingredient-gene-path" network was constructed. WXKL markedly decreased the incidence of arrhythmia in the MI pig model (P < 0.05). The QT interval was significantly shortened, and the heart rate was slowed down in the WXKL group compared with the model group (P < 0.05). In addition, the expression of sodium channel protein type 5 subunit alpha (SCN5A) and beta-2 adrenergic receptor (ADRB2) was downregulated, while muscarinic acetylcholine receptor M2 (CHRM2) was upregulated in the WXKL group (P < 0.05). In conclusion, WXKL may shorten the QT interval and slow down the heart rate by downregulating SCN5A and ADRB2 and upregulating CHRM2 during MI. These findings provide novel insight into molecular mechanisms of WXKL in reducing the incidence of ventricular arrhythmia.

Group II muscarinic acetylcholine receptors attenuate hepatic injury via Nrf2/ARE pathway.

Parasympathetic nervous system dysfunction is common in patients with liver disease. We have previously shown that muscarinic acetylcholine receptors (mAchRs) play an important role in the regulation of hepatic fibrosis and that the receptor agonists and antagonists affect hepatocyte proliferation. However, little is known about the impact of the different mAchR subtypes and associated signaling pathways on liver injury. Here, we treated the human liver cell line HL7702 with 10 mmol/L carbon tetrachloride (CCL4) to induce hepatocyte damage. We found that CCL4 treatment increased the protein levels of group I mAchRs (M1, M3, M5) but reduced the expression of group II mAchRs (M2, M4) and activated the Nrf2/ARE and MAPK signaling pathways. Although overexpression of M1, M3, or M5 led to hepatocyte damage with an intact Nrf2/ARE pathway, overexpression of M2 or M4 increased, and siRNA-mediated knockdown of either M2 or M4 decreased the protein levels of Nrf2 and its downstream target genes. Moreover, CCL4 treatment increased serum ALT levels more significantly, but only induced slight changes in the expression of mAchRs, NQO1 and HO1, while reducing the expression of M2 and M4 in liver tissues of Nrf2-/- mice compared to wild type mice. Our findings suggest that group II mAchRs, M2 and M4, activate the Nrf2/ARE signaling pathway, which regulates the expression of M2 and M4, to protect the liver from CCL4-induced injury.

M2 Receptor Activation Counteracts the Glioblastoma Cancer Stem Cell Response to Hypoxia Condition.

Glioblastoma multiforme (GBM) is the most malignant brain tumor. Hypoxic condition is a predominant feature of the GBM contributing to tumor growth and resistance to conventional therapies. Hence, the identification of drugs able to impair GBM malignancy and aggressiveness is considered of great clinical relevance. Previously, we demonstrated that the activation of M2 muscarinic receptors, through the agonist arecaidine propargyl ester (Ape), arrests cell proliferation in GBM cancer stem cells (GSCs). In the present work, we have characterized the response of GSCs to hypoxic condition showing an upregulation of hypoxia-inducible factors and factors involved in the regulation of GSCs survival and proliferation. Ape treatment in hypoxic conditions is however able to inhibit cell cycle progression, causing a significant increase of aberrant mitosis with consequent decreased cell survival. Additionally, qRT-PCR analysis suggest that Ape downregulates the expression of stemness markers and miR-210 levels, one of the main regulators of the responses to hypoxic condition in different tumor types. Our data demonstrate that Ape impairs the GSCs proliferation and survival also in hypoxic condition, negatively modulating the adaptive response of GSCs to hypoxia.

Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Pharmacogenetics of tardive dyskinesia in schizophrenia: The role of CHRM1 and CHRM2 muscarinic receptors.

Objectives: Acetylcholine M (muscarinic) receptors are possibly involved in tardive dyskinesia (TD). The authors tried to verify this hypothesis by testing for possible associations between two muscarinic receptor genes (CHRM1 and CHRM2) polymorphisms and TD in patients with schizophrenia.Methods: A total of 472 patients with schizophrenia were recruited. TD was assessed cross-sectionally using the Abnormal Involuntary Movement Scale. Fourteen allelic variants of CHRM1 and CHRM2 were genotyped using Applied Biosystems amplifiers (USA) and the MassARRAY System by Agena Bioscience.Results: The prevalence of the rs1824024*GG genotype of the CHRM2 gene was lower in TD patients compared to the group without it (χ2 = 6.035, p = 0.049). This suggested that this genotype has a protective effect for the development of TD (OR = 0.4, 95% CI: 0.19-0.88). When age, gender, duration of schizophrenia and dosage of antipsychotic treatment were added as covariates in regression analysis, the results did not reach statistical significance.Conclusions: This study did identify associations between CHRM2 variations and TD; the results of logistic regression analysis with covariates suggest that the association is, however, likely to be secondary to other concomitant factors.

Participation of acetylcholine and its receptors in the contractility of inflamed porcine uterus.

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10-5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10-5 M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10-5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10-5 M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10-7, 10-6 M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10-7 M) and ENDO/MYO (ACh: 10-5 M) than in the SAL group. MR2 or MR3 antagonists and ACh (10-5 M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10-6, 10-5 M) than in other groups. Both antagonists and ACh (10-5 M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.

Endocytic sorting and downregulation of the M2 acetylcholine receptor is regulated by ubiquitin and the ESCRT complex.

Cholinergic dysfunction plays a critical role in a number of disease states, and the loss of functional muscarinic acetylcholine receptors plays a key role in disease pathogenesis. Therefore, preventing receptor downregulation would maintain functional receptor number, and be predicted to alleviate symptoms. However, the molecular mechanism(s) underlying muscarinic receptor downregulation are currently unknown. Here we demonstrate that the M2 muscarinic receptor undergoes rapid lysosomal proteolysis, and this lysosomal trafficking is facilitated by ubiquitination of the receptor. Importantly, we show that this trafficking is driven specifically by ESCRT mediated involution. Critically, we provide evidence that disruption of this process leads to a re-routing of the trafficking of the M2 receptor away from the lysosome and into recycling pathway, and eventually back to the plasma membrane. This study is the first to identify the process by which the M2 muscarinic acetylcholine receptor undergoes endocytic sorting, and critically reveals a regulatory checkpoint that represents a target to pharmacologically increase the number of functional muscarinic receptors within the central nervous system.