Muscarinic Modulation of Synaptic Transmission and Short-Term Plasticity in the Dorsal and Ventral Hippocampus.

Muscarinic neurotransmission is fundamentally involved in supporting several brain functions by modulating flow of information in brain neural circuits including the hippocampus which displays a remarkable functional segregation along its longitudinal axis. However, how muscarinic neuromodulation contributes to the functional segregation along the hippocampus remains unclear. In this study we show that the nonselective muscarinic receptor agonist carbachol similarly suppresses basal synaptic transmission in the dorsal and ventral CA1 hippocampal field, in a concentration-depended manner. Furthermore, using a ten-pulse stimulation train of varying frequency we found that carbachol changes the frequency filtering properties more in ventral than dorsal hippocampus by facilitating synaptic inputs at a wide range of input frequencies in the ventral compared with dorsal hippocampus. Using the M2 receptor antagonist gallamine and the M4 receptor antagonist tropicamide, we found that M2 receptors are involved in controlling basal synaptic transmission and short-term synaptic plasticity (STSP) in the ventral but not the dorsal hippocampus, while M4 receptors participate in modulating basal synaptic transmission and STSP in both segments of the hippocampus. These results were corroborated by the higher protein expression levels of M2 receptors in the ventral compared with dorsal hippocampus. We conclude that muscarinic transmission modulates excitatory synaptic transmission and short-term synaptic plasticity along the entire rat hippocampus by acting through M4 receptors and recruiting M2 receptors only in the ventral hippocampus. Furthermore, M4 receptors appear to exert a permissive role on the actions of M2 receptors on STSP in the ventral hippocampus. This dorsoventral differentiation of muscarinic modulation is expected to have important implications in information processing along the endogenous hippocampal circuitry.

Maternal diabetes decreases the expression of α2-adrenergic and M2 muscarinic receptors in the visual cortex of male rat neonates.

AIMS: This study investigates the impact of maternal diabetes on the expression of α2-adrenergic and M2 muscarinic receptors in the primary visual cortex of male offspring born to diabetic rats. MAIN METHODS: In adult female rats, a single dose of intraperitoneal streptozotocin (STZ) was used to induce diabetes (Diabetic group). Diabetes was controlled with insulin in the Insulin-treated group. Female rats in the control group received normal saline instead of STZ. Male newborns were euthanized at P0, P7, and P14, and the expression of α2-adrenergic and M2 muscarinic receptors in the primary visual cortex was determined using immunohistochemistry (IHC). KEY FINDINGS: The study showed that α2-adrenergic and M2 muscarinic receptors were significantly suppressed in all layers of the primary visual cortex of male neonates born to diabetic rats at P0, P7, and P14 compared to the control group. The highest expression was for the Con group at P14 and the lowest one was in the Dia group at P0 for both receptors. The insulin treatment in diabetic mothers modulated the expression of these receptors to normal levels in their newborns. SIGNIFICANCE: The results demonstrate maternal diabetes decreases the expression of α2-adrenergic and M2 muscarinic receptors in the primary visual cortex of male offspring born to diabetic rats. Insulin treatment can offset these effects of diabetes.

M2 Muscarinic Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.

BACKGROUND: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8). METHODS: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9. RESULTS: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists. CONCLUSIONS: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

Analysis of Signal Transduction Pathways Downstream M2 Receptor Activation: Effects on Schwann Cell Migration and Morphology.

BACKGROUND: Schwann cells (SCs) express cholinergic receptors, suggesting a role of cholinergic signaling in the control of SC proliferation, differentiation and/or myelination. Our previous studies largely demonstrated that the pharmacological activation of the M2 muscarinic receptor subtype caused an inhibition of cell proliferation and promoted the expression of pro-myelinating differentiation genes. In order to elucidate the molecular signaling activated downstream the M2 receptor activation, in the present study we investigated the signal transduction pathways activated by the M2 orthosteric agonist arecaidine propargyl ester (APE) in SCs. METHODS: Using Western blot we analyzed some components of the noncanonical pathways involving ß1-arrestin and PI3K/AKT/mTORC1 signaling. A wound healing assay was used to evaluate SC migration. RESULTS: Our results demonstrated that M2 receptor activation negatively modulated the PI3K/Akt/mTORC1 axis, possibly through ß1-arrestin downregulation. The involvement of the mTORC1 complex was also supported by the decreased expression of its specific target p-p70 S6KThr389. Then, we also analyzed the expression of p-AMPKαthr172, a negative regulator of myelination that resulted in reduced levels after M2 agonist treatment. The analysis of cell migration and morphology allowed us to demonstrate that M2 receptor activation caused an arrest of SC migration and modified cell morphology probably by the modulation of ß1-arrestin/cofilin-1 and PKCα expression, respectively. CONCLUSIONS: The data obtained demonstrated that M2 receptor activation in addition to the canonical Gi protein-coupled pathway modulates noncanonical pathways involving the mTORC1 complex and other kinases whose activation may contribute to the inhibition of SC proliferation and migration and address SC differentiation.

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.

Impact of autoantibodies against the M2-muscarinic acetylcholine receptor on clinical outcomes in peripartum cardiomyopathy patients with standard treatment.

OBJECTIVES: To evaluate the impact of autoantibodies against the M2-muscarinic receptor (anti-M2-R) on the clinical outcomes of patients receiving the standard treatment for peripartum cardiomyopathy (PPCM). METHODS: A total of 107 PPCM patients who received standard heart failure (HF) treatment between January 1998 and June 2020 were enrolled in this study. According to anti-M2-R reactivity, they were classified into negative (n = 59) and positive (n = 48) groups, denoted as the anti-M2-R (-) and anti-M2-R (+) groups. Echocardiography, 6-min walk distance, serum digoxin concentration (SDC), and routine laboratory tests were performed regularly for 2 years. The all-cause mortality, cardiovascular mortality, and rehospitalisation rate for HF were compared between the two groups. RESULTS: A total of 103 patients were included in the final data analysis, with 46 in the anti-M2-R (+) group and 57 in the anti-M2-R (-) group. Heart rate was lower in the anti-M2-R (+) group than in the anti-M2-R (-) group at the baseline (102.7 ± 6.1 bpm vs. 96.0 ± 6.4 bpm, p < 0.001). The initial SDC was higher in the anti-M2-R (+) group than in the anti-M2-R (-) group with the same dosage of digoxin (1.25 ± 0.45 vs. 0.78 ± 0.24 ng/mL, p < 0.001). The dosages of metoprolol and digoxin were higher in the anti-M2-R (-) patients than in the anti-M2-R (+) patients (38.8 ± 4.6 mg b.i.d. vs. 27.8 ± 5.3 mg b.i.d., p < 0.0001, respectively, for metoprolol; 0.12 ± 0.02 mg/day vs. 0.08 ± 0.04 mg/day, p < 0.0001, respectively, for digoxin). Furthermore, there was a greater improvement in cardiac function in the anti-M2-R (-) patients than in the anti-M2-R (+) patients. Multivariate analysis identified negativity for anti-M2-R as the independent predictor for the improvement of cardiac function. Rehospitalisation for HF was lower in the anti-M2-R (-) group, but all-cause mortality and cardiovascular mortality were the same. CONCLUSIONS: There were no differences in all-cause mortality or cardiovascular mortality between the two groups. Rehospitalisation rate for HF decreased in the anti-M2-R (-) group. This difference may be related to the regulation of the autonomic nervous system by anti-M2-R.

M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

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.

M2 muscarinic autoantibodies and thyroid hormone promote susceptibility to atrial fibrillation and sinus tachycardia in an autoimmune rabbit model.

NEW FINDINGS: What is the central question of this study? Do autoantibodies to the M2 muscarinic receptor (M2R-AAbs) have the potential to facilitate specific sustained tachyarrhythmias in the presence of thyroxine (T4 ) in rabbits? What is the main finding and its importance? The M2R-AAb and T4 jointly destabilized the electrophysiological properties, thus promoting the occurrence of atrial and sinus tachyarrhythmias in rabbits. These findings provide a practical basis for understanding the pathophysiological role of M2R-AAb alone and with T4 in arrhythmia induction and might provide an innovative option for treatment of Graves' disease with rhythm disturbance. ABSTRACT: Activating autoantibodies toward the ß1/2 -adrenergic receptors (ß1/2AR-AAbs) and M2 muscarinic receptor (M2R-AAbs) are present in a high proportion of patients with Graves' disease. We previously demonstrated that ß1/2AR-AAbs with or without the presence of M2R-AAbs in combination with excessive thyroxine (T4 ) increased the induction of sustained tachyarrhythmias in an autoimmune rabbit model. However, the separate role of M2R-AAbs and their interaction with T4 are not clear. The aim of this study was to investigate the impact of M2R-AAbs and T4 on the induction of cardiac arrhythmias in a similar rabbit model. Ten New Zealand White rabbits were randomly divided into two groups. In group A (n = 6), the rabbits were immunized with the second extracellular loop peptide of M2R and subjected to 2 weeks of T4 treatment. In group B (n = 4), the rabbits were treated only with T4 for 2 weeks. After induction of general anaesthesia, rabbits were subjected to an electrophysiological study at 0 (pre-immune), 6 (post-immune) and 8 weeks (post-immune+T4 treatment) in group A and at 0 (baseline) and 8 weeks (T4 treatment) in group B. Each rabbit served as its own control. In group A, high levels and activity of M2R-AAbs were detected in all immunized animals. Thyroxine in combination with immunization significantly increased induction of sustained sinus tachycardia and atrial fibrillation in comparison to the pre-immune state. In group B, T4 predominantly induced sustained sinus tachycardia. This study demonstrated that M2R-AAbs and T4 jointly increased the susceptibility to both sinus and atrial tachyarrhythmias. The data supported the pathophysiological role of M2R-AAbs in hyperthyroidism-associated supraventricular tachyarrhythmias.

Activating autoantibodies against G protein-coupled receptors in narcolepsy type 1.

STUDY OBJECTIVES: Narcolepsy type 1 is a rare hypersomnia of central origin, which is caused by loss of hypothalamic neurons that produce the neuropeptides hypocretin-1 and -2. Hypocretin-containing nerve terminals are found in areas known to play a central role in autonomic control and in pain signaling. Cholinergic M2 receptors are found in brain areas involved with the occurrence of hallucinations and cataplexy. In addition to classical symptoms of narcolepsy, the patients suffer frequently from autonomic dysfunction, chronic pain, and hypnagogic/hypnopompic hallucinations. We aimed to test whether narcolepsy type 1 patients have autoantibodies against autonomic ß2 adrenergic receptor, M2 muscarinic receptors, or nociception receptors. METHODS: We tested the serum of ten narcolepsy type 1 patients (five female) for activating ß2 adrenergic receptor autoantibodies, M2 muscarinic receptor autoantibodies, and nociception receptor autoantibodies. RESULTS: Ten of ten patients were positive for muscarinic M2 receptor autoantibodies (P < 0.001), 9/10 were positive for autoantibodies against nociception receptors (P < 0.001), and 5/10 were positive for ß2 adrenergic receptor autoantibodies (P < 0.001). CONCLUSIONS: Narcolepsy type 1 patients harbored activating autoantibodies against M2 muscarinic receptors, nociception receptors, and ß2 adrenergic receptors. M2 receptor autoantibodies may be related to the occurrence of cataplexy and, moreover, hallucinations in narcolepsy since they are found in the same brain areas that are involved with these symptoms. The occurrence of nociception receptor autoantibodies strengthens the association between narcolepsy type 1 and pain. The connection between narcolepsy type 1, autonomic complaints, and the presumed cardiovascular morbidity might be associated with the occurrence of ß2 adrenergic receptor autoantibodies. On the other hand, the presence of the autoantibodies may be secondary to the destruction of the hypocretin pathways.

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.

The relationship between ß1 -adrenergic and M2 -muscarinic receptor autoantibodies and hypertrophic cardiomyopathy.

NEW FINDINGS: What is the central question of this study? The concentrations of ß1 -adrenergic receptor and M2 -muscarinic receptor autoantibodies in hypertrophic cardiomyopathy (HCM) patients and the relationship between the cardiac autoantibodies and clinical manifestations of HCM have rarely been reported. What is the main finding and its importance? We found that the concentrations of the two autoantibodies in HCM patients were significantly higher than those in control subjects. Furthermore, we found that the concentrations of the two autoantibodies could reflect myocardial injury and diastolic dysfunction in HCM patients to some extent and might be involved in the occurrence of arrhythmia. These findings might be valuable in exploration of the mechanisms of occurrence and progression of HCM. ABSTRACT: Increasing attention is being given to the role of immunological mechanisms in the development of heart failure. The purpose of this study was to investigate the concentration of serum ß1 -adrenergic receptor autoantibody (ß1 -AAb) and M2 -muscarinic receptor autoantibody (M2 -AAb) in patients with hypertrophic cardiomyopathy (HCM), and the relationship between ß1 -AAb, M2 -AAb and clinical indices. One hundred and thirty-four patients with HCM were recruited consecutively into the HCM group. Forty healthy subjects were assigned as the normal controls (NCs). Serum samples were collected to measure the concentrations of ß1 -AAb and M2 -AAb by enzyme-linked immunosorbent assay. The clinical data of HCM patients were collected. The serum concentrations of ß1 -AAb and M2 -AAb of HCM patients were significantly higher than those of NCs. In HCM patients, those with a left atrial diameter ≥50 mm or moderate-to-severe mitral regurgitation had significantly higher concentrations of the two autoantibodies. Patients with a history of syncope had higher concentrations of ß1 -AAb. Female patients and patients with a family history of sudden cardiac death or atrial fibrillation had higher concentrations of M2 -AAb. Maximal wall thickness, interventricular septum thickness and resting left ventricular outflow tract gradient were positively correlated with log ß1 -AAb or log M2 -AAb in HCM patients. In conclusion, the serum concentrations of ß1 -AAb and M2 -AAb of HCM patients were significantly higher than those of NCs. Being female, syncope, a family history of sudden death, atrial fibrillation, left atrial diameter ≥50 mm, moderate-to-severe mitral regurgitation, maximal wall thickness, interventricular septum thickness and resting left ventricular outflow tract gradient may affect the concentrations of the two autoantibodies.

Association of autoantibodies against the M2-muscarinic receptor with long-term outcomes in peripartum cardiomyopathy patients: A 5-year prospective study.

BACKGROUND: Peripartum cardiomyopathy (PPCM) is characterized by heart failure. Our previous study found that autoantibodies against the M2-muscarinic receptor (anti-M2-R) are increased in PPCM patients. We aimed to evaluate the association of anti-M2-R on prognosis of PPCM patients with standard treatment. METHODS: Synthetic peptides corresponding to the M2 receptor served as the target antigens in an enzyme-linked immunosorbent assay experiment. They were used to screen the sera of 80 PPCM patients, who were separated into anti-M2-R-negative and positive groups according to their anti-M2-R reactivity. Clinical assessment and echocardiography examination were performed at baseline and after 5 years with a standard treatment regimen. The endpoint events were compared after 5 years of follow-up. RESULTS: There were 76 PPCM patients who completed the final data analysis, including 36 in the anti-M2-R (+) group and 40 in the anti-M2-R (-) group. Both groups showed improvement in the left ventricular end-diastolic and end-systolic dimensions and the ejection fraction with standard treatment regimens for 5 years (all p<0.001). Patients in the anti-M2-R (-) group had greater tolerance and were more rapidly titrated to metoprolol, and they had better improvement in cardiac function than patients in the anti-M2-R (+) group (p<0.05). Patients in the anti-M2-R (-) group had a marked decrease in re-hospitalization (p<0.05), but not in all-cause mortality or cardiovascular mortality. Being positive for anti-M2-R increased the risk of PPCM (OR=4.7, 95% CI 1.8-12.2, p=0.002). CONCLUSIONS: PPCM patients, especially anti-M2-R (-) patients, have a relatively better prognosis than other patients. We posit that the presence of anti-M2-R may be involved in the pathogenesis of PPCM.

Contribution of M1 and M2 muscarinic receptor subtypes to convulsions in fasted mice treated with scopolamine and given food.

Treatment of fasted mice and rats with the nonselective muscarinic antagonist, scopolamine or atropine, causes convulsions after food intake. This study evaluated the effect of fasting on the expression of M1 and M2 muscarinic receptors in the brain regions, the relationship between receptor expression and seizure stages, and the muscarinic receptor subtype which plays a role in the occurrence of convulsions. Mice were grouped as allowed to eat ad lib (fed) and deprived of food for 24h (fasted). Fasted animals developed convulsions after being treated with scopolamine (60%) or the selective M1 receptor antagonist pirenzepine (10mg/kg; 20% and 60mg/kg; 70%) and given food. Fasting increased expression of M1 receptors in the frontal cortex and M2 receptors in the hippocampus, but produced no change in the expression of both receptors in the amygdaloid complex. Food intake after fasting decreased M1 receptor expression in the frontal cortex and M1 and M2 receptor expression in the hippocampus. Seizure severity was uncorrelated with muscarinic receptor expression in the brain regions. Taken together, these findings provide evidence for the role of M1 muscarinic receptor antagonism and fasting-induced increases in M1 and M2 expression possible underlying mechanism in the occurrence of convulsions in fasted animals.

Autoradiography of 3H-pirenzepine and 3H-AFDX-384 in Mouse Brain Regions: Possible Insights into M1, M2, and M4 Muscarinic Receptors Distribution.

Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target. 3H-Pirenzepine is commonly believed to label predominantly M1MR, 3H-AFDX-384 is considered as M2MR selective ligand. Here we performed series of autoradiographies with 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with 3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of 3H-AFDX-384 toward M2MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M2MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of 3H-AFDX-384 binding sites, respectively, are represented by M4MR and M2MR constitute only a minor portion. In cortex and hippocampus, 3H-AFDX-384 labels almost similar amounts of M2MR and M4MR alongside significant amounts of non-M2/non-M4MR. In cortex, the proportion of 3H-AFDX-384 binding sites attributable to M2MR can be increased by blocking M4MR with MT3 toxin without affecting non-M4MR. PD102807, which is considered as a highly selective M4MR antagonist failed to improve the discrimination of M2MR. Autoradiography with 3H-QNB showed genotype specific loss of binding sites. IN CONCLUSION: while 3H-pirenzepine showed the high selectivity toward M1MR, 3H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.

FRET-based nanobiosensor for detection of scopolamine in hairy root extraction of Atropa belladonna.

A simple, sensitive, selective, and rapid optical nanobiosensor based on FRET was designed to detect tropane alkaloids as anti-cholinergic agents in natural and transgenic hairy roots extracts of Atropa belladonna. To achieve that, conjugation of tioglycolyic acid capped cadmium telluride quantum Dots, M2 muscarinic receptor (Cd/Te QDs-M2R) and conjugation of scopolamine-rhodamine123 (Sc-Rho123) were performed. More specifically, proportional amounts of M2 muscarinic receptor and quantum dots (QDs) were conjugated while scopolamine (as a tropane alkaloid) and rhodamine123 were also combined and these moieties functioned as donor and acceptor pairs, respectively. The system response was linear over the range of 0.01-4µmolL-1 of scopolamine hydrochloride concentration with a detection limit of 0.001µmolL-1. The developed nanobiosensor was successfully used for in vitro recognition of scopolamine as an anti-cholinergic agent in the investigated plant extracts. In addition, Agrobacterium rhizogenesis mediated gene transfer technique was employed to generate hairy roots and to enhance the production of tropane alkaloids in the studied medicinal plant.

Association of ß1-Adrenergic, M2-Muscarinic Receptor Autoantibody with Occurrence and Development of Nonvalvular Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is the most common sustained arrhythmia and is independently associated with increased risk of stroke and death. Although the exact mechanisms of AF are not completely elucidated, a large number of evidences demonstrate that autoimmunity may play an important role in the initiation, the progression, and the maintenance of AF. In this study, we aimed to compare anti-ß1-adrenergic receptor autoantibody (anti-ß1-R) and anti-M2-muscarinic receptor autoantibody (anti-M2-R) levels between nonvalvular AF patients and healthy control subjects. METHODS: The levels of serum anti-ß1-R, antinuclear antibodies, and anti-M2-R were measured in both groups by enzyme-linked immunosorbent assay (ELISA). High-sensitivity C-reactive protein (hs-CRP) and interleukin (IL)-6 concentration were measured, respectively, by immunoturbidimetry and chemiluminescence. RESULTS: Anti-ß1-R and anti-M2-R levels were significantly higher in patients with nonvalvular AF than in healthy controls (anti-ß1-R 221.11 [132.38-291.69] ng/mL vs 198.14 [125.70-278.40] ng/mL, P < 0.01; anti-M2-R 271.81 [144.99-378.20] ng/mL vs 235.01 [121.53-358.99] ng/mL, P < 0.01). Compared with the control group, the serum levels of IL-6 and hs-CRP were higher in the nonvalvular AF group (IL-6 19.65 ± 5.6 pg/mL vs 6.79 ± 1.09 pg/mL, hs-CRP 6.03 ± 1.35 mg/L vs 2.73 ± 0.63 mg/L, P < 0.05). Antinuclear antibody (ANA) levels were similar between two groups (ANA 10.55 [1.86-271.8] U/mL vs 10.49 [1.303-161.7] U/mL, P > 0.05). The baseline value of serum anti-ß1-R (odds ratio [OR]: 13.176, P < 0.001), anti-M2-R (OR: 4.41, P < 0.001), IL-6 (OR: 6.126, P < 0.05) levels, and left atrial diameter (OR: 5.781, P < 0.05) were independent predictors of nonvalvular AF by multivariable analyses. CONCLUSION: We found a significant association between circulating serum anti-ß1-R, anti-M2-R, IL-6 levels, and nonvalvular AF. We presume that the autoimmunity and inflammation might take part in electrical remodeling and structural remodeling of left atrium.

Inactivation of M2 AChR/NF-κB signaling axis reverses epithelial-mesenchymal transition (EMT) and suppresses migration and invasion in non-small cell lung cancer (NSCLC).

Non-neuronal cholinergic system is involved in lung physiology and lung cancer. However, the biochemical events downstream acetylcholine (ACh) receptor activation leading to carcinogenesis and tumor progression are not fully understood. Our previous work has shown that non-neuronal ACh acts as an autoparacrine growth factor to stimulate cell proliferation and promote epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) via activation of M2 muscarinic receptor (M2R). The aim of the present study was to delineate the underlying mechanisms linking M2R and lung tumor progression, which may provide potential therapeutic targets to delay lung cancer progression. Inhibition of M2R by antagonist or siRNA suppresses NSCLC cell migratory and invasive capacities, reverses EMT and simultaneously inactivates PI3K/Akt, MAPK ERK and NF-κB p65. On the other hand, M2R activation stimulates NSCLC migration and invasion and promotes EMT via NF-κB p65 activation. Moreover, NF-κB p65 activation induced by M2R activation was partially inhibited by either Akt or ERK inhibitor. Taken together, these results demonstrated for the first time that NF-κB p65 activation is essential in NSCLC progression associated with non-neuronal cholinergic system. Our data suggest that M2R/ERK/Akt/NF-κB axis could be a potential target for NSCLC treatment.