The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.
BACKGROUND: Tulbaghia violacea Harv. (Alliaceae) is used to treat various ailments, including hypertension (HTN) in South Africa. This study aims to evaluate the contributions of muscarinic receptors and changes in plasma aldosterone levels to its anti-hypertensive effect. METHODS: In the acute experiments, methanol leaf extracts (MLE) of T. violacea (30 - 120 mg/kg), muscarine (0.16 -10 ug/kg), and atropine (0.02 - 20.48 mg/kg), and/or the vehicle (dimethylsulfoxide (DMSO) and normal saline (NS)) were respectively and randomly administered intravenously in a group of spontaneously hypertensive (SHR) weighing 300 to 350g and aged less than 5 months. Subsequently, T. violacea (60 mg/kg) or muscarine (2.5 ug/kg) was infused into eight SHRs, 20 minutes after atropine (5.12 mg/kg) pre-treatment. In the chronic (21 days) experiments, the SHRs were randomly divided into three groups, and given the vehicle (0.2 ml/day of DMSO and NS), T. violacea (60 mg/kg/day) and captopril (10 mg/kg/day) respectively into the peritoneum, to investigate their effects on blood pressure (BP), heart rate (HR), and plasma aldosterone levels. Systolic BP and HR were measured using tail-cuff plethysmography during the intervention. BP and HR were measured via a pressure transducer connecting the femoral artery and the Powerlab at the end of each intervention in the acute experiment; and on day 22 in the chronic experiment. RESULTS: In the acute experiments, T. violacea, muscarine, and atropine significantly (p < 0.05) reduced BP dose-dependently. T. violacea and muscarine produced dose-dependent decreases in HR, while the effect of atropine on HR varied. After atropine pre-treatment, dose-dependent increases in BP and HR were observed with T. violacea; while the BP and HR effects of muscarine were nullified. In the chronic experiments, the T. violacea-treated and captropril-treated groups had signicantly lower levels of aldosterone in plasma when compared to vehicle-treated group. Compared to the vehicle-treated group, significant reduction in BP was only seen in the captopril-treated group; while no difference in HR was observed among the groups. CONCLUSION: The results obtained in this study suggest that stimulation of the muscarinic receptors and a reduction in plasma aldosterone levels contribute to the anti-hypertesive effect of T. violacea.
The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR-selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.
Effect of auricular acupuncture on gastrointestinal motility and its relationship with vagal activity.
- Acupuncture in medicine : journal of the British Medical Acupuncture Society
- Published over 5 years ago
BACKGROUND: Vagus nerve stimulation is capable of regulating autonomic nerve function. In Traditional Chinese Medicine, the effect of auricular acupuncture (AA) is mediated by the vagus. This study was designed to investigate the effect of AA on gastrointestinal (GI) motility and the relationship of this effect with the vagus nerve. METHODS: 50 rats were divided into five groups for observation of the effects of different types of acupuncture and influencing factors: control, AA, somatic acupuncture (SA), atropine and atropine+AA. The acupuncture points used for AA were ST (Stomach) and SI (Small intestine), while the acupuncture point used for SA was ST36. Electroacupuncture was performed for 15 min. A model of reduced GI motility was established using ethanol, and GI transit rate was used to measure GI motility. Heart rate variability (HRV) and the effect of atropine administration were investigated to study the relationship between AA and vagal activity. RESULTS: The GI transit rate increased in both the AA and SA groups compared with control, and no significant difference was found between their effects. In addition, after atropine administration, AA was found to be ineffective in influencing the GI transit rate. In the HRV analysis, no significant differences were found in the absolute low frequency normalised units, high frequency normalised units or the low frequency/high frequency component ratio in the AA or SA groups compared with control. After administration of atropine AA still had no effect on HRV. CONCLUSIONS: The function of AA in improving GI motility is similar to that of SA, and this effect can be blocked by the presence of atropine, indicating that this effect is regulated by the vagus. However, HRV did not reflect the acupuncture-induced changes in vagal nerve function.
Datura stramonium is an herbaceous annual plant. All parts of the plant contain tropane alkaloids such as atropine and scopolamine. We report the case of a 22-year-old man admitted to a general hospital for visual and aural hallucinations. One week after his admission, as the hallucinations remained, the patient was transferred to a psychiatric hospital. Neither blood nor urine was conserved during his hospitalization, so a hair analysis was requested in order to identify a possible consumption of a Datura seed infusion.
Circulating leucocytes express muscarinic (m) and nicotinic (n) receptors and synthesize acetylcholine (ACh) regulating various cell functions. Leucocytes from patients with cystic fibrosis contain less ACh; therefore it was tested whether the regulation of cellular functions like migration differed from healthy volunteers.
Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.
Twelve homology models of the human M2 muscarinic receptor using different sets of templates have been designed using the Prime program or the modeller program and compared to crystallographic structure (PDB:3UON). The best models were obtained using single template of the closest published structure, the M3 muscarinic receptor (PDB:4DAJ). Adding more (structurally distant) templates led to worse models. Data document a key role of the template in homology modeling. The models differ substantially. The quality checks built into the programs do not correlate with the RMSDs to the crystallographic structure and cannot be used to select the best model. Re-docking of the antagonists present in crystallographic structure and relative binding energy estimation by calculating MM/GBSA in Prime and the binding energy function in YASARA suggested it could be possible to evaluate the quality of the orthosteric binding site based on the prediction of relative binding energies. Although estimation of relative binding energies distinguishes between relatively good and bad models it does not indicate the best one. On the other hand, visual inspection of the models for known features and knowledge-based analysis of the intramolecular interactions allows an experimenter to select overall best models manually.
Vasomotor Effects of Acetylcholine, Bradykinin, Noradrenaline, 5-Hydroxytryptamine, Histamine and Angiotensin II on the Mouse Basilar Artery
- The Journal of veterinary medical science / the Japanese Society of Veterinary Science
- Published about 4 years ago
We investigated the responsiveness of the mouse basilar artery to acetylcholine (ACh), bradykinin (BK), noradrenaline (NA), 5-hydroxytryptamine (5-HT), histamine (His) and angiotensin (Ang) II in order to characterize the related receptor subtypes in vitro. ACh and BK induced endothelium-dependent relaxation of precontracted arteries with U-46619 (a thromboxane A2 analogue). Atropine (a non-selective muscarinic receptor antagonist) and Nω-nitro-L-arginine (a NO synthase inhibitor, L-NNA) shifted the concentration-response curve for ACh to the right, whereas pirenzepine, methoctramine and pFHHSiD (muscarinic M1, M2 and M3 antagonists, respectively) had no significant effect. L-NNA and HOE140 (a B2 antagonist) shifted the concentration-response curve for BK to the right, whereas Des-Arg(9)-[Leu(8)]-BK (a B1 antagonist) and indomethacin (a COX inhibitor) had no significant effect. NA failed to produce any vasomotor action. His and Ang II induced concentration-dependent contraction. Diphenhydramine (a H1 antagonist) shifted the concentration-response curve for His to the right, whereas cimetidine (a H2 antagonist) had no significant effect. Losartan (an AT1 antagonist) shifted the concentration-response curve for Ang II to the right, whereas PD123319 (an AT2 antagonist) had no significant effect. These results suggest that the H1 and AT1 receptor subtypes might play an important role in arterial contraction, whereas muscarinic receptor subtypes other than M1, M2 and M3, and B2 receptors on the endothelium, might modify these contractions to relaxations.