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.

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.

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）.

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.

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.

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.

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.

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.

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.

Enzyme-linked Immunosorbent Assay for Detection of Anti-idiotype Antibodies to Antibodies to Ligand of Nicotinic Acetylcholine Receptor in Sera of Patients with Myasthenia Gravis

Anti-bungarotoxin anti-serum,which has the internal image of nicotinicacetylcholine receptor,was used as a tool to measure anti-idiotypic antibodies toantibodies to Iigand of nicotinic acctylcholine receptor in scra from 81 patients withmyasthenia gravis.Enzyme-linked immunosorbcnt assay was adopted.Thc positive ratewas 46.9%(38/81).The specific cross inhibitory test with nicotinic acetylcholinereceptor was positive.Anti-idiotype antibodies to antibodies to ligand of nicotinicacetylcholine receptor in sera of different types of myasthenia gravis patients classified ac-cording to modified Osserman’s standard and myasthenia gravis patients with or withoutthymoma were comparcd in this study and the role of anti-idiotype antibodies toantibodies to Iigand of nicotinic acctylcholinc receptor in the immunity of myasthcniagravis and the possibility of thcrapeutic use of anti-idiotype antibodies arc discussed.

Effect Factors in Synthesis of Nicotinic Acid by Electrooxidation of 3-Picoline

In an electrolytic cell with a proton exchange membrane, nicotinic acid was synthesized at the PbO2 anode. The relationship between the current density and the potential at different concentrations of 3-picoline and sulfuric acid as well as at different temperatures was studied with polarization curves. The effects of the concentrations of sulfuric acid and 3-picoline, the anode potential and the reaction temperature on the selectivity and the current efficiency were explored. The optimum conditions were determined by orthogonal experiments. Under optimum conditions the selectivity and the current efficiency for the synthesis of nicotinic acid might reach 89% and 65%, respectively. The, concentrations of 3-picoline and nicotinic acid were analyzed with high performance liquid chromatography and the product was characterized with elemental analysis, chromatography-mass spectrometry and IR spectrometry.

Effect of soman,sarin and VX on the nicotinic acetylcholine receptor

The highly specific ligand of the N-acetylcholine receptor（N-AChR）,alpha-bungarotoxin,was used to determine the effect of soman,sarin and VX onN-AChR of the diaphragm and extensor digitorum Iongus muscle of mice andrats.The effects of the three anti-cholinesterase agents on N-AChR weredifferent.Sarin did not act directly on N-AChR and cause a change in the numberof N-AChR.VX decreased the binding sites of the receptor by binding with N-AChRdirectly.The LD50was 0.054mg/kg in mousse.Soman increased the binding sites,e.g.1～1.5 LD50soman increased the number of N-AChR of mouse diaphragm by 25%.The peak increaseof N-AChR was reached 0.5 h after poisoning and could last 96h.The receptornumber was still 22% higher than that of the control on the fourth day aftersoman poisoning in rats.Soman mainly increased the number of extrasynapticN-AChR,leading to the enhancement of sensitivity of cholinergic effectors toacetylcholine（ACh）,which is similar to the hypersensitiveness resulting fromdenervation.These findings are of significance in probing the receptor mecha-nisms and treatment of soman poisoning.

The Catalysis of NAD^+, NADP^+ and Nicotinic Amide for Methanol Electrooxidation at Platinum Electrode

A group of liquid catalysts composed of nicotinic amide functioning on the anode of DMFC were investigated at a Pt eleetrode, which were nicotinic amide, nicotinamide adenine dinucleotide （ NAD^＋ ） and its phosphate （ NAD （ P ） ^＋ ）. The kinetics of methanol anode oxidation in the three reaction systems was compared by measuring poteatiodynarnic current-potential curves and AC impedances. The experimeatal results show that the dynamic behavior of methanol oxidation at a Pt electrode has been changed with adding the three substances. The influence of temperature on the catalysis of these coenzymes and nicotinic amicle was discussed by comparing the AC impedances spectra of methanol oxidation at differeat temperatures.

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.