Atropine can induce autophagy independent of the M3 muscarinic acetylcholine receptor

Background: No other effects of atropine other than as an antagonist of muscarinic acetylcholine receptor (mAChR) have been found. Methods: In this study, human kidneyepithelial cells were treated with different physiological regulators. Results: Subsequently, it was found that atropine could significantly induce autophagy as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm ofhost cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and increased p62/SQSTM1 indicatedincomplete autophagy. In addition, atropine induced autophagosome levels in a dose-dependent manner within a certain concentration range in human kidney epithelial cells. In atropine-treated mouse skeletal muscle cells containing nicotinic acetylcholinereceptors and rat cardiac muscle cells containing mAchR, atropine induced autophagy in mouse skeletal muscle cells but not in rat cardiac muscle cells. Furthermore, atropine did not induce autophagy in tissue cells containing mAchR in vivo but did in tissue cells not containing mAchR. Conclusion: This study expands the application and understanding of atropine’s action mechanism in the field of medicine.

Anisodine hydrobromide alleviates oxidative stress caused by hypoxia/reoxygenation in human cerebral microvascular endothelial cells predominantly via inhibition of muscarinic acetylcholine receptor 4

Background:Anisodine hydrobromide(AT3),an anti-cholinergic agent,could be delivered to the brain across the blood-brain barrier and has been used clinically for the treatment of cerebral ischemia/reperfusion injury.Endothelial dysfunction can be caused by hypoxia/reoxygenation(H/R)via oxidative stress and metabolic alterations.The present study investigated whether AT3 regulates the production of nitric oxide(NO)and reactive oxygen species(ROS),and the HIF-1αpathway via regulation of muscarinic acetylcholine receptors(mAChRs)in brain microvascular endothelial cells after H/R exposure.Methods:Under H/R conditions,hCMEC/D3 cerebral microvascular endothelial cells were treated with AT3.Specific inhibitors of M2-and M4-mAChRs were used to explore the mechanism by which AT3 influences oxidative stress in endothelial cells.Then,mAChRs expression was detected by western blotting and NO production was detected by Greiss reaction.The intracellular ROS level was measured using DCFH-DA probes.The expression of hypoxia-inducible transcription factor 1α(HIF-1α)was also detected.Results:While H/R induced the expression of M2-and M4-mAChRs,AT3 suppressed the H/R-upregulated M2-and M4-mAChRs.H/R also induced the production of NO,ROS,and apoptosis.AT3 and M4-mAChR inhibitors inhibited the H/R-induced production of NO and ROS and apoptosis.HIF-1αwas induced by H/R,but was suppressed by AT3.Conclusion:Thus,the in vitro evidence shows that AT3 protects against H/R injury in cerebral microvascular endothelial cells via inhibition of HIF-1α,NO and ROS,predominantly through the downregulation of M4-mAChR.The findings offer novel understandings regarding AT3-mediated attenuation of endothelial cell apoptosis and cerebral ischemia/reperfusion injury.

Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

Distribution of Like-muscarinic Acetylcholine Receptor M2 in the Brain of Three Castes of Polyrhachis vicina

The cholinergic system plays an important role in the central nervous system of insects and is closely related to the complex behavior of insects. The immunohistochemical technique was performed to detect the expression of like-muscarinic acetylcholine receptor M2 in the brain of three castes of Polyrhachis vicina. A positive expression of like-muscarinic acetylcholine receptor M2 was observed in the mushroom body, central body and antennal lobes of the ant brain; but there is great diversity in their location and intensity among worker, queen and male ants. It is speculated that like-muscafinic acetylcholine receptor M2 plays a critical role in the central nervous system, in terms of projecting visual information and olfactory information into the protocerebrum and integrating many inputs.

AduoLa Fuzhenglin Down-regulates Microwave-induced Expression of β_1-adrenergic Receptor and Muscarinic Type 2 Acetylcholine Receptor in Myocardial Cells of Rats

This paper is aimed to study the effect of ADL on expression of ~z-AR and Mz-AchR in myocardial cells of rats exposed to microwave radiation. Immunohistochemistry, Western blot and image analysis were used to detect the expression of ~I-AR and Mz-AchR in myocardial cells at 7 and 14 d after microwave exposure. The results show that the expression level was higher in microwave exposure group and 0.75 g/（kg.d） ADL group than in sham operation group and significantly lower in 1.5 and 3.0 g/（kg.d） ADL groups than in microwave group. So we have a conclusion that the expression of I^z-AR and Mz-AchR is down-regulated in myocardial cells of rats exposed to microwave radiation. ADL can protect rats against microwave-induced heart tissue injury.

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles.The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction,the nicotinic acetylcholine receptor.Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse.This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis.The review also covers the application of a powerful biophysical technique,superresolution optical microscopy,to image the nicotinic receptor in live cells and follow its motional dynamics.The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor,as observed in myasthenia gravis.

Insensitive Acetylcholine Receptor Conferring Resistance of Plutella xylostella to Nereistoxin Insecticides

The combinative rate measurement of （3-[I125] iodotyrosyl） α-bungarotoxin was applied in the analysis of the relation between nerve acetylcholine receptor and three types of insecticide resistance in diamondback moth, Plutella xylostella （L.）. In the dimehypo-resistant strain and in the cartap-resistant strain, the nerve acetylcholine receptor showed the remarkable insensitivity to dimehypo and cartap, of which the binding rate to ligand was approximately 66 and 60%, respectively, of the susceptible strain. The sensitivity to deltamethrin in the deltamethrin-resistant strain did not show visible change. These results indicated that the decline in the sensitivity of nerve acetylcholine receptor to insecticide might be a potential mechanism to nereistoxin insecticides resistance in the diamondback moth.

Targeting α7 nicotinic acetylcholine receptors: a future potential for neuroprotection from traumatic brain injury

Traumatic brain injury （TBI） poses a significant socioeconomic burden in the world. The long lasting consequences in cognitive impairments are often underreported and its mechanisms are unclear. In this perspective, cholinergic dysfunction and thera-peutic strategy targeting this will be reviewed. Novel agents that can target specific subtype of acetylcholine receptors have been developed over the recent years and are at various stages of development, which include AR-R 17779, GTS-21, SSR- 180711A, AR-R17779, and PNU-282987. A detailed review on this topic has been previously published （Shin and Dixon, 2015）.

Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion

The present study found expressions of a7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibdllary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein la, RANTES, interleukin-1β, intedeukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid 13 protein 1-42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1-42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated a7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1-42.

Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer＇s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which influence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to persistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in persistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer＇s disease.

Adult celiac disease with acetylcholine receptor antibody positive myasthenia gravis

Celiac disease has been associated with some autoimmune disorders. A 40-year-old competitive strongman with celiac disease responded to a gluten-free diet, but developed profound and generalized motor weakness with acetylcholine receptor antibody positive myasthenia gravis, a disorder reported to occur in about 1 in 5000. This possible relationship between myasthenia gravis and celiac disease was further explored in serological studies. Frozen stored serum samples from 23 acetylcholine receptor antibody positive myasthenia gravis patients with no intestinal symptoms were used to screen for celiac disease. Both endomysial and tissue transglutaminase antibodies were examined. One of 23 (or, about 4.3%) was positive for both IgA-endomysial and IgA tissue transglutaminase antibodies. Endoscopic studies subsequently showed duodenal mucosal scalloping and biopsies confirmed the histopathological changes of celiac disease. Celiac disease and myasthenia gravis may occur together more often than is currently appreciated. The presence of motor weakness in celiac disease may be a clue to occult myasthenia gravis, even in the absence of intestinal symptoms.

“Warming yang and invigorating qi” acupuncture alters acetylcholine receptor expression in the neuromuscular junction of rats with experimental autoimmune myasthenia gravis

Myasthenia gravis is an autoimmune disorder in which antibodies have been shown to form against the nicotinic acetylcholine nicotinic postsynaptic receptors located at the neuromuscular junction.＂Warming yang and invigorating qi＂ acupuncture treatment has been shown to reduce serum inflammatory cytokine expression and increase transforming growth factor beta expression in rats with experimental autoimmune myasthenia gravis.However,few studies have addressed the effects of this type of acupuncture on the acetylcholine receptors at the neuromuscular junction.Here,we used confocal laser scanning microscopy to examine the area and density of immunoreactivity for an antibody to the nicotinic acetylcholine receptor at the neuromuscular junction in the phrenic nerve of rats with experimental autoimmune myasthenia gravis following ＂warming yang and invigorating qi＂ acupuncture therapy.Needles were inserted at acupressure points Shousanli（LI10）,Zusanli（ST36）,Pishu（BL20）,and Shenshu（BL23） once daily for 7 consecutive days.The treatment was repeated after 1 day of rest.We found that area and the integrated optical density of the immunoreactivity for the acetylcholine receptor at the neuromuscular junction of the phrenic nerve was significantly increased following acupuncture treatment.This outcome of the acupuncture therapy was similar to that of the cholinesterase inhibitor pyridostigmine bromide.These findings suggest that ＂warming yang and invigorating qi＂ acupuncture treatment increases acetylcholine receptor expression at the neuromuscular junction in a rat model of autoimmune myasthenia gravis.

M3 Muscarinic Acetylcholine Receptor Antagonist Darifenacin Protects against Pulmonary Fibrosis through ERK/NF-κB/miR-21 Pathway

Idiopathic pulmonary fibrosis is an untreatable lethal lung disease, which is related to the aberrant proliferation of fibroblasts. M3 muscarinic acetylcholine receptor (M3-mAChR) activation exerts proliferative effect on various kinds of cells. However, whether M3-mAChR inhibition has a protective effect on pulmonary fibrosis remains unexplored. A rat model of pulmonary fibrosis was established by intratracheal instillation of bleomycin. Darifenacin was used to block M3-mAChR. Histological changes were observed using Masson’s Trichrome and hematoxylin and eosin (HE) staining. Hydroxyproline was measured by Hydroxyproline detection kit. Transforming growth factor β1 (TGF-β1) and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). In vitro, pulmonary fibroblasts were isolated from lungs of neonatal rat. After treatment, the cell viability, Hydroxyproline level was measured by MTT and Hydroxyproline detection kit respectively. The expression level of extracellular signal-regulated kinase (ERK), nuclear factor kappa-B (N-NF-κB), and microRNA-21 (miR-21) was detected by western blot or quantitative real-time PCR (qRT-PCR). Darifenacin relieved the fibrotic effects provoked by bleomycin. The expression level of hydroxyproline, TGF-β1 and TNF-α level was all downregulated after darifenacin treatment. In lung fibroblasts, darifenacin decreased cell viability and hydroxyproline level induced by bleomycin. Besides, phosphorylation-ERK and nuclear N-NF-κB protein level was downregulated, as well as miR-21 level. M3-mAChR antagonist darifenacin attenuates bleomycin-induced pulmonary fibrosis in rats, which may relate to the ERK/NF-κB/miRNA-21 signaling pathway.

Computational Determination of the Binding Mode of α-Conotoxin to Nicotinic Acetylcholine Receptor

Abstract Conotoxins belong to the large families of disulfide-rich peptide toxins from cone snail venom, and can act on a broad spectrum of ion channels and receptors. They are classified into different subtypes based on their targets. The a-conotoxins selectively inhibit the current of the nicotinic acetylcholine receptors. Because of their unique selectivity towards distinct nAChR subtypes, a-conotoxins become valuable tools in nAChR study. In addition to the X-ray structures of a-conotoxins in complex with acetyleholine-binding protein, a homolog of the nAChR ligand-binding domain, the high-resolution crystal structures of the extracellular domain of the al and a9 subunits are also obtained. Such structures not only revealed the details of the configuration of nAChR, but also provided higher sequence identity templates for modeling the binding modes of a-conotoxins to nAChR. This mini-review summarizes recent modeling studies for the determination of the binding modes of a-conotoxins to nAChR. As there are not crystal structures of the nAChR in complex with conotoxins, computational modeling in combination of mutagenesis data is expected to reveal the molecular recognition mechanisms that govern the interactions between a-conotoxins and nAChR at molecular level. An accurate determination of the binding modes of a-conotoxins on AChRs allows rational design of a-conotoxin analogues with improved potency or selectivity to nAChRs.

Effect of differential rearing environments on nicotine-stimulated locomotor activity and nicotinic acetylcholine receptor subtypes

OBJECTIVE Individuals vary in sensitivity to the behavioral effects of nicotine,resulting in differences in their vulnerability to addiction.The role of rearing environment in determining individual sensitivity to nicotine is unclear.The neuropharmacological mechanisms mediating the effect of rearing environment on the actions of nicotine are also understood.Thus,the contribution of rearing environment in determining the sensitivity to the locomotor effects of nicotine and regulating α4β2*-and α7-nicotinic acetylcholine(n ACh) receptor expressionwas determined in rats reared in isolated(IC) or enriched(EC) conditions.METHODS To measure locomotor activity,adolescent rats(postnatal day 21-51)were injected with saline(1 mL·kg^(-1)) or nicotine(0.3 mg·kg^(-1)) subcutaneously,then placed in chamberswhere ambulatory activity was monitored for 30-min by computer for 14 daily sessions.α4β2*-andα7-n ACh receptor expression in the mesolimbic dopamine pathway was determined by quantitative autoradiography of [125 I]-epibatidine and [125 I]-bungarotoxinbinding,respectively,in 16 μmol·L^(-1) coronal sections.Values for receptor expression in fmol are ±s of 8 brains and compared by two-tailed,unpaired t-test with P<0.05 considered significant.RESULTS EC-rats are similarly sensitive as IC-rats to the locomotor effects of nicotine.[125 I]-epibatidine binding in the ventral tegmental area of EC-rats was reduced(2.8±0.3 fmo L) compared to IC-rats(4.0±0.4 fmo L);there was no difference in the nucleus accumbens.There was no difference between EC-and IC-rats in α7-n ACh receptor expression in the mesolimbic dopamine pathway.CONCLUSION Rearing environment differentially regulates n ACh receptor subtypes in EC and IC rats.These data suggest regulation of n ACh receptors by environmental factors may be a mechanism for the protective effect of enrichment against altered sensitivity to nicotine in genetically vulnerable individuals.The characterization of these mechanisms will aid in development of novel pharmacological tools mimicking the protection afforded by environmental enrichment in nicotine-sensitive individuals.

Nanoscale interactions between the nicotinic acetylcholine receptor and cholesterol

Cholesterol is a major lipid in biological membranes.It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels.The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine(nAChR)contain linear sequences of amino acids with the capacity to recognize cholesterol.These cholesterol consensus domains have been designated as“CARC”and its mirror sequence“CRAC”.CARC preferentially occurs in the exoplasmic-facing membrane leaflet,and CRAC,in the cytoplasmic-facing hemilayer.Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems,where they recognize cholesterol,and in prokaryotic homologues in bacteria,where they recognize hopanoids.This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions,probably contributing to the stability of membrane-embedded protein domains.Structural studies from our laboratory using superresolution optical microscopy(“nanoscopy”)have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR.The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility(diffusion)in the plane of the membrane.

Structural Dynamics of Amyloid β Peptide Binding to Acetylcholine Receptor and Virtual Screening for Effective Inhibitors

The interaction between Amyloid β(Aβ) peptide and acetylcholine receptor is the key for our understanding of how Aβ fragments block the ion channels within the synapses and thus induce Alzheimer’s disease.Here,molecular docking and molecular dynamics(MD)simulations were performed for the structural dynamics of the docking complex consisting of Aβ and α7-n ACh R(α7 nicotinic acetylcholine receptor),and the inter-molecular interactions between ligand and receptor were revealed.The results show that Aβ_(25-35) is bound toα7-n ACh R through hydrogen bonds and complementary shape,and the Aβ_(25-35) fragments would easily assemble in the ion channel of α7-n ACh R,then block the ion transfer process and induce neuronal apoptosis.The simulated amide-I band of Aβ_(25-35) in the complex is located at 1650.5 cm^(-1),indicating the backbone of Aβ_(25-35) tends to present random coil conformation,which is consistent with the result obtained from cluster analysis.Currently existing drugs were used as templates for virtual screening,eight new drugs were designed and semi-flexible docking was performed for their performance.The results show that,the interactions between new drugs and α7-n ACh R are strong enough to inhibit the aggregation of Aβ_(25-35) fragments in the ion channel,and also be of great potential in the treatment of Alzheimer’s disease.

Nicotinic Acetylcholine Receptor Gene Family of the Pea Aphid,Acyrthosiphon pisum

The nicotinic acetylcholine receptors （nAchRs） are cholinergic receptors that form ligand-gated ion channels by ifve subunits in insect and vertebrate nervous systems. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Here, we identiifed and cloned 11 candidate nAChR subunit genes in Acyrthosiphon pisum using genome-based bioinformatics combined modern molecular techniques. Most A. pisum nAChRs including α1, α2, α3, α4, α6, α8, and β1 show highly sequence identities with the counterparts of other insects examined. Expression proifles analysis showed that all subunit genes were expressed in adult head. At least two subunits have alternative splicing that obviously increase A. pisum nicotinic receptor diversity. This study will be invaluable for exploring the molecular mechanisms of neonicotinoid-like insecticides in sucking pests, and for ultimately establishing the screening platform of novel insecticides.

Clinical Measurement of Antibodies against Acetylcholine Receptor (AchR),SOD and LPO in Patients with Myasthenia Gravis (MG)before and after Thymectomy

The antibodies against acetylcholine receptor AchR and levels of SOD and LPO were measured in 11 patients with myasthenia gravis (MG),and the results were compared with normal controls and patients with diseases other than MG.The results showed that the antibodies against AchR were higher as compared with other groups before and after operation. The post-operative level of antibodies was obviously lower than the pre-operative value. An slight increase in SOD and significant decrease in mean value of LPO after surgery were noted. The possible mechanism was discussed.

Muscarinic Acetylcholine Receptor Subtype Expression in Type Vestibular Hair Cells of Guinea Pigs

Recent studies have demonstrated that five subtypes （M1-M5） of muscarinic acetylcholine receptor （mAChR） are expressed in the vestibular periphery. However, the exact cellular location of the mAChRs is not clear. In this study, we investigated whether there is the expression of M1-M5 muscarinic receptor mRNA in isolated type Ⅱ vestibular hair cells of guinea pig by using single-cell RT-PCR. In vestibular end-organ, cDNA of the expected size was obtained by RT-PCR. Moreover, mRNA was identified by RT-PCR from individually isolated type Ⅱ vestibular hair cells （single-cell RT-PCR）. Sequence analysis confirmed that the products were M1-M5 mAChR. These results dem-onstrated that M1-M5 mAChR was expressed in the typeⅡvestibular hair cells of the guinea pig, which lends further support for the role of M1-M5 mAChR as a mediator of efferent cholinergic signalling pathway in vestibular hair cells.