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.

Exploitation of the nicotinic anti-inflammatory pathway for the treatment of epithelial inflammatory diseases

Discoveries in the first few years of the 21st century have led to an understanding of important interactions between the nervous system and the inflammatory response at the molecular level, most notably the acetylcholine （ACh）- triggered,α7-nicotinic acetylcholine receptor （α7nAChR）- dependent nicotinic antinflammatory pathway. Studies using the α7nAChR agonist, nicotine, for the treatment of mucosal inflammation have been undertaken but the efficacy of nicotine as a treatment for inflammatory bowel diseases remains debatable. Further understanding of the nicotinic anti-inflammatory pathway and other endogenous anti-inflammatory mechanisms is required in order to develop refined and specific therapeutic strategies for the treatment of a number of inflammatory diseases and conditions, including periodontitis, psoriasis, sarcoidosis, and ulcerative colitis.

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.

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.

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.

Neuroprotective and anti-inflammatory effects of a therapy combining agonists of nicotinic α7 and σ1 receptors in a rat model of Parkinson’s disease

To date there is no treatment able to stop or slow down the loss of dopaminergic neurons that characterizes Parkinson’s disease.It was recently observed in a rodent model of Alzheimer’s disease that the interaction between the α7 subtype of nicotinic acetylcholine receptor(α7-nAChR)and sigma-1 receptor(σ1-R)could exert neuroprotective effects through the modulation of neuroinflammation which is one of the key components of the pathophysiology of Parkinson’s disease.In this context,the aim of the present study was to assess the effects of the concomitant administration of N-(3R)-1-azabicyclo[2.2.2]oct-3-yl-furo[2,3-c]pyridine-5-carboxamide(PHA)543613 as an α7-nAChR agonist and 2-(4-morpholinethyl)1-phenylcyclohexanecarboxylate(PRE)-084 as aσ1-R agonist in a well-characterized 6-hydroxydopamine rat model of Parkinson’s disease.The animals received either vehicle separately or the dual therapy PHA/PRE once a day until day 14 postlesion.Although no effect was noticed in the amphetamine-induced rotation test,our data has shown that the PHA/PRE treatment induced partial protection of the dopaminergic neurons(15-20%),assessed by the dopamine transporter density in the striatum and immunoreactive tyrosine hydroxylase in the substantia nigra.Furthermore,this dual therapy reduced the degree of glial activation consecutive to the 6-hydroxydopamine lesion,i.e,the 18 kDa translocation protein density and glial fibrillary acidic protein staining in the striatum,and the CD11b and glial fibrillary acidic protein staining in the substantia nigra.Hence,this study reports for the first time that concomitant activation of α7-nAChR andσ1-R can provide a partial recovery of the nigro-striatal dopaminergic neurons through the modulation of microglial activation.The study was approved by the Regional Ethics Committee(CEEA Val de Loire n°19)validated this protocol(Authorization N°00434.02)on May 15,2014.

Alpha-7 nicotinic acetylcholine receptor agonist treatment in a rat model of Huntington's disease and involvement of heme oxygenase-1

Neuroinflammation is a common element involved in the pathophysiology of neurodegenerative diseases.We recently reported that repeated alpha-7 nicotinic acetylcholine receptor（α7 n ACh R） activations by a potent agonist such as PHA 543613 in quinolinic acid-injured rats exhibited protective effects on neurons.To further investigate the underlying mechanism,we established rat models of early-stage Huntington＇s disease by injection of quinolinic acid into the right striatum and then intraperitoneally injected 12 mg/kg PHA 543613 or sterile water,twice a day during 4 days.Western blot assay results showed that the expression of heme oxygenase-1（HO-1）,the key component of the cholinergic anti-inflammatory pathway,in the right striatum of rat models of Huntington＇s disease subjected to intraperitoneal injection of PHA 543613 for 4 days was significantly increased compared to the control rats receiving intraperitoneal injection of sterile water,and that the increase in HO-1 expression was independent of change in α7 n ACh R expression.These findings suggest that HO-1 expression is unrelated to α7 n ACh R density and the increase in HO-1 expression likely contributes to α7 n ACh R activation-related neuroprotective effect in early-stage Huntington＇s disease.

Therapeutic potential of α7 nicotinic receptor agonists to regulate neuroinflammation in neurodegenerative diseases

Neurodegenerative diseases, such as Alzheimer＇s, Parkinson＇s and Huntington＇s diseases, are all character- ized by a component of innate immunity called neuroinflammation. Neuronal loss and neuroinflammation are two phenomena closely linked. Hence, the neuroinflammation is a relevant target for the management of the neurodegenerative diseases given that, to date, there is no treatment to stop neuronal loss. Several studies have investigated the potential effects of activators of alpha 7 nicotinic acetylcholine receptors in animal models of neurodegenerative diseases. These receptors are widely distributed in the central nervous system. After activation, they seem to mediate the cholinergic anti-inflammatory pathway in the brain. This anti-inflammatory pathway, first described in periphery, regulates activation of microglial cells considered as the resident macrophage population of the central nervous system. In this article, we shortly review the agonists of the alpha 7 nicotinic acetylcholine receptors that have been evaluated in vivo and we focused on the selective positive allosteric modulators of these receptors. These compounds represent a key element to enhance receptor activity only in the presence of the endogenous agonist.

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）.

Reactive Extraction of Nicotinic Acid with Trialkylamine in n-Octanol

The distributions of nicotinic acid (NA) between water and trialkylamine (N235) dissolved in n-octanol was studied. The complexes of N235 and NA were investigated by Fourier transformation infrared spectrometry to deduce the reaction mechanism. It was found that N235/n-octanol was an efficient extractant for extracting nicotinic acid. The favorable operation conditions were equilibrium aqueous pH 4.2 to 5.5 and initial N235 concentration<0.42 mol·L-1 . The reaction processes included the reaction between neutral N235 and neutral NA and the reaction between protonated N235 and anionic NA. Based on the mass action law and some assumptions, an expression for distribution coefficient D was proposed. The apparent extraction equilibrium constants were calculated by fitting the experimental data and the results were satisfactory.

Synthesis of Nicotinic Acid by Potassium Dichromate Oxidation

Nicotinic acid was synthesized from 3 methylpyridine with potassium dichromate as the oxidant. The reaction conditions, the product separation and analysis were studied. The results indicate that under the certain reaction conditions, the yield of nicotinic acid can reach 67.4%, the conversion can reach 99.7% and the selectivity can be as high as 99.8%. The final product has a high purity.

Nicotinic cholinergic receptors in esophagus:Early alteration during carcinogenesis and prognostic value

AIM: To compare expression of nicotinic cholinergic receptors(CHRNs) in healthy and squamous cell carcinoma-affected esophagus and determine the prognostic value.METHODS: We performed RT-q PCR to measure the expression of CHRNs in 44 esophageal samples from healthy individuals and in matched normal surrounding mucosa, and in tumors from 28 patientsdiagnosed with esophageal squamous cell carcinoma(ESCC). Next, we performed correlation analysis for the detected expression of these receptors with the habits and clinico-pathological characteristics of all study participants. In order to investigate the possible correlations between the expression of the different CHRN subunits in both healthy esophagus and tissues from ESCC patients, correlation matrices were generated. Subsequently, we evaluated whether the detected alterations in expression of the various CHRNs could precede histopathological modifications during the esophageal carcinogenic processes by using receiver operating characteristic curve analysis. Finally, we evaluated the impact of CHRNA5 and CHRNA7 expression on overall survival by using multivariate analysis.RESULTS: CHRNA3, CHRNA5, CHRNA7 and CHRNB4, but not CHRNA1, CHRNA4, CHRNA9 or CHRNA10, were found to be expressed in normal(healthy) esophageal mucosa. In ESCC, CHRNA5 and CHRNA7 were overexpressed as compared with patient-matched surrounding non-tumor mucosa(ESCC-adjacent mucosa; P < 0.0001 and P = 0.0091, respectively). Positive correlations were observed between CHRNA3 and CHRNB4 expression in all samples analyzed. Additionally, CHRNB4 was found to be differentially expressed in the healthy esophagus and the normalappearing ESCC-adjacent mucosa, allowing for distinguishment between these tissues with a sensitivity of 75.86% and a specificity of 78.95%(P = 0.0002). Finally, CHRNA5 expression was identified as an independent prognostic factor in ESCC; patients with high CHRNA5 expression showed an increased overall survival, in comparison with those with low expression. The corresponding age- and tumor stage-adjusted hazard ratio was 0.2684(95%CI: 0.075-0.97, P = 0.0448).CONCLUSION: Expression of CHRNs is homogeneous along healthy esophagus and deregulated in ESCC, suggesting a pathogenic role for these receptors in ESCC development and progression.

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.

Preparation of Nicotinic Acid from Oxidation of 3-Picoline with Oxygen Under Catalysis of T(o-Cl)PPMn

The oxidation of 3-picoline to nicotinic acid took place efficiently in an ethanol solution with 02 as the oxidant under the catalysis of T（o-Cl）PPMn at 40--150 ℃ and 0.5--3.0 MPa oxygen pressure. The influences of temperature, oxygen pressure, reaction time, concentration of 3-picoline, concentration of sodium hydroxide, and concentration of T（o-Cl）PPMn catalyst, etc. on the production of nicotinic acid were investigated. The results show that T（o-Cl）PPMn presented excellent catalytic activity in the oxidation Of 3-pieoiine to nicotinic acid and the yield of nicotinic acid varied greatly with the reaction temperature, oxygen pressure, T（o-Cl）PPMn concentration, etc.

Nicotinic acid induces apoptosis of glioma cells via the calciumdependent endoplasmic reticulum stress pathway

Malignant glioma is one of the most common and deadly tumors in the central nervous system while developing effective treatments for this devastating disease remains a challenge.Previously,we demonstrated that the vitamin nicotinic acid(NA)inhibits glioma invasion.Here,we show that high-dose NA induces apoptosis of malignant glioma cells in vitro and in vivo.In cultured U251 glioma cells treated with NA,we detected ER stress that was likely caused by elevated intracellular calcium levels.The elevated calcium can be attributed to the activation of TRPV1,a cation channel that has been implicated in cutaneous flushing caused by NA administration.Our data further suggested that NA-induced apoptosis is mediated by the calcium-dependent proteases called calpains,whose activities are drastically upregulated by NA.NA-induced apoptosis of U251 cells can be attenuated by blocking calpain activity or knocking down TRPV1.These results reveal a novel function of NA in regulating glioma cell apoptosis via the calcium-dependent ER stress pathway and imply a potential application of NA for the treatment of malignant glioma.

Activation of α7 nicotinic acetylcholine receptor protects against oxidant stress damage through reducing vascular peroxidase-1 in a JNK signaling-dependent manner in endothelial cells

Aim Alpha7 nicotinic acetylcholine receptor （α7nAChR）, a subtype of nAChR regulating neurotrans- mission in central nervous system, is an essential regulator of cholinergic antiinflammatory pathway in periphery. The present study was to determine the effects of activation of α7nAChR on oxidant stress-induced injury in endo- thelial cells. Methods Cultured human umbilical vein endothelial cells were treated with H202 （400 μmol · L^-1） or H202plus PNU-282987 （ 10 μmol · L^-1 ）. Cell viability and membrane integrity were measured. AnnexinV ＋ PI assay, immunoblotting of bcl-2, bax and cleaved caspase-3, and immunofluorescence of apoptosis inducing factor （AIF） were performed to evaluate apoptosis. Protein expression of vascular peroxidase-1 （ VPO-1 ） and phosphor- JNK were measured by immunoblotting. Results Activation of α7nAChR by a selective agonist PNU-282987 pre-vented H202-indced decrease of cell viability and increase of lactate dehydrogenase release. Activation of α7nAChR markedly reduced cell apoptosis and intracellular oxidative stress level. Moreover, activation of α7nAChR reduced H2 02 -induced VPO-1 protein upregulation and JNK1/2 phosphorylation. The inhibitory effect of α7nAChR activa- tion on VPO-1 was blocked by JNK inhibitor SP600125. In addition, pretreatment of α7nAChR antagonist methyl- lycaconitine blocked the cytoprotective effect of PNU-282987. Conclusion These results provide the first evidence that activation of α7nAChR protects against oxidant stress-induced damage by suppressing VPO-1 in a JNK signa- ling pathway-dependent manner in endothelial cells.

Role of α3 nicotinic acetylcholine receptor subunit in the inflammatory responses of atherosclerosis

Aim The expression of α3 subunit of nicotinic acetylcholine receptor （α3-nAChR） has been demonstra- ted in aorta, adipocyte and macrophage. The objective of the present study was to verify the regulatory roles of α3- nAChR in the inflammatory responses of atherosclerosis. Methods The inflammatory indicators were detected in mouse macrophage, adipocytes and mouse aortic endothelial cells （MAECs） after the α3-nAChR was antagonized or after the α3-nAChR gene was silenced. Meanwhile, atherogenesis was induced in the apolipoprotein E knock-out （ ApoE^ -/- ） mice after fed with an atherogenic high-fat diet for 7 weeks. Results In MAECs, the lipopolysaccha- ride （LPS）-stimulated secretions of the adhesion molecules and inflammatory cytokines were significantly enhanced （30%± 80% ） after pretreatment with α-Conotoxin MII （an antagonist for α3-nAChR） or after knock-down with α3-nAChR gene. In adipocytes, the knock-down of α3 gene promoted the generations of the proin？ ammatory adi- pokines or cytokines but decreased the production of adiponectin, an anti-inflammatory adipokine, by 29.29 ± 9.43%. In macrophage silenced with α3-nAChR gene, the M1 （classical） activation was predominantly stimula- ted, whereas the M2 （alternative） activation was suppressed. In addition, the amount of the atherosclerotic lesions and the infiltration of the M1 type activated macrophages into the arterial wall were markedly elevated in the α- Conotoxin MII-treated ApoE -/- mice. Conclusion The α3-nAChR may play a pivotal role in regulating the atherogenesis through influencing the inflammatory responses of ECs, macrophages and adipocytes. The mecha- nisms involve the regulations of multiple cell signaling pathways.

Differentiation of the Agonists and Antagonists of the α7 Nicotinic Acetylcholine Receptor

Theα7 nicotinic acetylcholine receptors(nAChRs)are widely expressed in the central and peripheral nervous systems and are important drug targets for the treatment of neurological diseases.However,differentiation of the agonists and antagonists of the nAChR is difficult.In this study we aimed to develop a reliable and efficient computational approach for differentiation of the agonists from the antagonists of the nAChR based on a systematical analysis of 123 ligands(87 agonists,12 partial agonists,and 24 antagonists)binding with the extracellular domain of theα7 n AChR chimera.Our results suggest that the ligand size and ligand binding affinity cannot differentiate the agonists from the antagonists of the nAChR.The ligand efficiency that considers both ligand binding affinity and size for the agonists is overall more left shifted in comparison to the antagonists,but the values of the ligand efficiency still cannot differentiate the agonists from the antagonists unless the values are either relatively high(more than-0.3 kcal mol^-1)or relatively low(less than-0.45 kcal mol^-1).Our results suggest that accurate prediction of the agonist or antagonist of the nAChR is challenging and the ligand innate configuration has to be considered as an extra for differentiation of the agonists from the antagonists of the nAChR.

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.