α4亚基Arg-188和Pro-198突变促进α4β2烟碱型乙酰胆碱受体开放

目的:探讨乙酰胆碱、尼古丁、伐尼克兰和金雀花碱4种激动剂与α4β2烟碱型乙酰胆碱受体(nAChRs)及其突变体相互作用的活性关键位点.方法:体外转录法制备野生型(wt)与突变型α4亚基cRNAs,注射入非洲爪蟾卵母细胞,应用电生理方法,测定4种激动剂对α4β2 nAChRs及其突变体的门控活性.结果:乙酰胆碱对wtα4...     

葡萄糖氧化酶诱导的氧化应激抑制α4β2烟碱型乙酰胆碱受体电流

目的探究葡萄糖氧化酶(GO)引起的氧化应激对α4β2烟碱型乙酰胆碱受体(nAChR)电流的影响。方法在HEK293T细胞共转染α4和β2质粒48 h后进行实验。分为对照组(control组)、3.5 k U/L的GO处理组(GO组)、3.5 k U/L的GO与450 k U/L的过氧化氢酶(CAT)共处理组(CAT组)。使用DCFH-DA荧光探针以及激光共聚焦显微镜来检测活性氧(ROS)的产生;采用全细胞膜片钳检测α4β2 nAChR的电流变化,给予1、10和30μmol/L ACh检测受体功能,给予10μmol/L ACh检测电流变化。结果全细胞膜片钳记录到呈ACh剂量依赖性的电流;GO处理HEK293T细胞1 h后可显著地增强细胞活性氧的水平(P0.001);对照组电流在10次给药中均保持稳定,GO组的电流在连续10次的ACh给药下逐渐降低(P0.01);CAT组反转GO引起的α4β2 nAChR电流下降(P0.01)。结论GO引起的氧化应激状态导致α4β2 nAChR电流逐渐下降,α4β2 nAChR电流的下降与ROS的增多密切相关。     

rL-RVG通过α7烟碱型乙酰胆碱受体诱导胃癌细胞系SGC凋亡的机制

目的探讨表达狂犬病毒疫苗糖蛋白的重组新城疫病毒(rL-RVG)通过乙酰胆碱受体诱导胃癌细胞凋亡的可能机制。方法将对数增殖期的胃癌细胞系SGC随机分成rL-RVG、新城疫病毒(NDV)组、PBS组,并同时分别设α7烟碱型乙酰胆碱受体(α7-nAChR)抑制剂(MLA)及激动剂(ACB)预处理组。病毒感染24 h后,CCK8法测定病毒对SGC增殖影响;蛋白印迹检测α7-nAChR、RVG、NDV;凋亡蛋白cleved-caspase-3、BAX/BCL-2;P-ERK、ERK的表达。免疫荧光法测定P-ERK表达。结果随着rL-RVG、NDV病毒液浓度增加,胃癌细胞活力下降,rL-RVG在10~(-2)稀释倍数时抗增殖作用显著(P0.05);病毒组凋亡相关蛋白cleved-caspase-3、BAX/BCL-2相对表达量较PBS组明显升高,且rL-RVG组较NDV组及PBS组升高明显,乙酰胆碱受体抑制剂预处理后rL-RVG组、NDV组及PBS组凋亡蛋白水平明显增高,胆碱受体激动剂预处理组凋亡蛋白表达水平明显下降(P0.05);P-ERK在病毒组表达下降,且rL-RVG组较NDV组更低,乙酰胆碱受体抑制剂、激动剂预处理后分别下降、上升(P0.05)。结论rL-RVG通过α7-nAChR诱导胃癌细胞系SGC凋亡的机制之一是抑制ERK通路。     

烟碱型乙酰胆碱受体α3和/或α5型在大鼠脑垂体中的表达:荧光双标免疫组织化学研究

为了探讨在大鼠脑垂体中,包含α3和 /或α5亚单位的烟碱型乙酰胆碱受体(N -AChR)是否存在,我们运用免疫荧光双标记技术对成年雄性大鼠脑垂体进行了研究。结果显示:N -AChR的α3和 /或α5亚单位样免疫反应阳性物质局限性地分布在神经垂体的催产素能神经纤维上,并一直延续到神经垂体的尾侧末端。     

尼古丁对多种干细胞中烟碱型乙酰胆碱受体表达的影响

目的:明确干细胞中烟碱型乙酰胆碱受体(nAChRs)亚基的表达以及尼古丁处理后受体改变的情况。方法:选择胚胎、胚胎样干细胞和成体干细胞作为探究对象。采用无饲养层细胞的方法培养人胚胎干细胞系H9和人诱导多能干细胞(IPSC);成体干细胞选择人脐带间充质干细胞(UCMSC)和人骨髓间充质干细胞(BMMSC)。4种细胞传代培养后分成对照组和处理组,处理组培养基中加入尼古丁,分别提取总RNA和蛋白。采用RT-PCR、RT-qPCR和Western blot检测nAChRs亚基的mRNA和蛋白表达水平,采用细胞免疫荧光染色检测受体亚基的表达位置。结果:RT-PCR和Western blot结果显示,H9细胞、IPSC和UCMSC中均检测到nAChRs的α2、α3、α4、α7、α9和β1亚基,而BMMSC仅α4、α7、α9和β1表达明显。尼古丁处理后,RT-qPCR结果显示α2在H9细胞和UCMSC中显著下调,而在IPSC中发生上调;β2在BMMSC和IPSC中显著上调,而在UCMSC中显著下调。结论:胚胎干细胞和成体干细胞中表达的nAChRs亚基不同,且分化潜能越高,表达种类越多。尼古丁对nAC...     

α7烟碱样乙酰胆碱受体调节干细胞功能的研究进展

α7烟碱型乙酰胆碱受体(alpha 7 nicotinic acetylcholine receptor,α7nACHR)作为一种神经递质受体,广泛存在于各种非神经组织,参与调节细胞增殖、分化及生存等功能。最新的研究发现：α7nACHR不仅在成熟的组织和器官,而且也在未分化的干/祖细胞中表达。作为一种重要的受体,具有调节干/祖细胞自我更新和分化的作用。本文就α7nACHR在干细胞中的表达及其作用特征,尤其与干细胞功能障碍相关疾病的关联机制进行了综述。期望以此了解α7nACHR在干细胞中的作用,并为以α7nACHR为靶点的新药研发及干细胞治疗提供可靠的依据。     

神经型烟碱乙酰胆碱受体α7亚单位基因多态性与精神分裂症的传递不平衡研究

目的 探讨神经型烟碱乙酰胆碱受体α7亚单位基因(neuronal nicotinic acetyleholine receptor α7 subunit gene,CHRNA7)多态性与精神分裂症的关系.方法 应用聚合酶链反应及聚丙烯酰胺凝胶芯片技术检测129个精神分裂症先证者核心家系CHRNA7基因的rs2337980、rs1909884、rs883473三个单核苷酸多态性,并采用基于单倍型的单倍型相对风险检验(haplotype relative risk,HHRR)、传递不平衡检验(transmission disequilibrium test,TDT)及单倍型分析进行统计.结果 (1)HHRR分析结果显示rs2337980位点精神分裂症患者组与虚拟对照组之间等位基因频率差异有统计学意义(P=0.017);(2)TDT分析发现,rs2337980位点与精神分裂症之间可能存在传递不平衡,杂合子父母过多的传递等位基因C给患病子女(P=0.021).(3)单倍型分析发现,rs2337980、rsl909884及rs2337980、rsl909884、rs883473组成的单倍型与精神分裂症有显著相关(总体P=0.034;glohal P=0.027),其中T-C,T-C-T两个单倍型与精神分裂症可能存在传递不平衡.结论 CHRNA7 基因多态性可能与精神分裂症存在关联,rs2337980的变异等位基因T可能是精神分裂症的保护性因子.     

用电压钳检测烟碱型乙酰胆碱受体导电孔道的重要位点

目的 利用双电极电压钳技术检测组成人烟碱型乙酰胆碱受体离子导电孔道的主要氨基酸位点.方法 用定点突变的方法将人alpha 7烟碱型乙酰胆碱受体(α7 nAChR)亚基跨膜区段M2的氨基酸(Leu248-Ala258)逐一突变为亲水性的丝氨酸,随后利用cDNA质粒体外转录获得cRNA,并将单个突变体的cRNA注射到非洲爪...     

横纹肌肉瘤胚胎型骨骼肌乙酰胆碱受体γ亚基mRNA表达的临床诊断价值

目的探讨胚胎型骨骼肌乙酰胆碱受体γ亚基(AChR-7)mRNA的表达在横纹肌肉瘤病理诊断中的价值。方法运用双重逆转录一聚合酶链反应(RT-PCR)在17例石蜡包埋横纹肌肉瘤[胚胎型(ERMS)9例,腺泡型(ARMS)6例,多形型(PRMS)2例]、20例非横纹肌源性肿瘤(低分化滑膜肉瘤6例,尤文／原始神经外胚层肿瘤6例,淋巴瘤6例,神经母细胞瘤2例)和3例正常肌肉中同时检测AChR-γ和α亚基(AChR-α)mRNA的表达并比较两者比值。结果17例横纹肌肉瘤均表达AChR-α和AChR-γ,且两者表达量的比值即AChR-α/AChR-α≥1,20例非横纹肌源性小圆细胞肿瘤无AChR-1的表达,1例正常肌肉AChR-α/AChR-α＜1。结论检测AChR-γmRNA的表达有助于横纹肌肉瘤的诊断和鉴别诊断。     

PDLLM5与α7烟碱乙酰胆碱受体的相互作用及作用位点的研究

目的研究支架蛋白PDLIM5与神经元α7烟碱乙酰胆碱受体(α7nAChR)间的相互作用及其作用位点。方法构建PDLIM5基因及其截短片段原核、真核重组表达载体和构建α7nAChR真核重组表达载体,并在大肠杆菌中表达与纯化相关蛋白。采用GST pull down及免疫共沉淀(CO-IP)方法检测PDLIM5与α7nAChR分子间相互作用及结合位点。结果 GST pull down和CO-IP方法检测结果显示PDLIM5与α7nAChR具有相互作用,且α7nAChR与PDLIM5中的PDZ结构域具有特异的相互作用,但不与PDLIM5中的LIM结构域发生反应。结论 PDLIM5通过其PDZ结构域介导同α7nAChR的相互作用。     

Properties of mutated murine alpha4beta2 nicotinic receptors linked to partial epilepsy

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.     

Monoclonal antibody to β2 subunit of neuronal nicotinic receptor depresses the postjunctional non-contractile Ca mobilization in the mouse diaphragm muscle

The involvement of subtypes of nicotinic acetylcholine receptor (nAChR) in the postjunctional non-contractile Ca²⁺ mobilization was investigated in mouse diaphragm muscles treated with an anticholinesterase, using monoclonal antibodies (mAbs) to nAChR subunits. mAb 210 (specific for 1 subunit of muscle nAChR) depressed contractile Ca²⁺ transients without affecting non-contractile Ca²⁺ transients. mAb 270 (specific for β2 subunit of neuronal nAChR) depressed only non-contractile Ca²⁺ transients. mAb 210 did not completely block the ACh-activated channel currents in flexor digitorum brevis muscle cells. The present findings indicate that the anti-β2 mAb 270-related subtype of nAChR may postsynaptically operate the non-contractile Ca²⁺ mobilization at the neuromuscular junction, suggesting the involvement of a subtype different from the usual muscle-type nAChR.     

不同β-肾上腺素能受体对低氧应激大鼠心脏舒缩功能的影响

目的:探讨不同β-肾上腺素能受体(β-adrenergic receptor,β-AR)在急性低氧应激中对大鼠左、右心室舒缩功能的影响。方法:健康雄性SD大鼠随机分为4组(n=7):对照组(control group)、非选择性β-肾上腺素能受体阻断剂普萘洛尔组(propranolol group)、选择性β_1-肾上腺素能受体阻断剂阿替洛尔组(atenolol group)和选择性β2-肾上腺素能受体阻断剂ICI 118,551组(ICI 118,551 group),各组大鼠分别在常氧(西宁,海拔2 260 m,20.9% O_2,79.1% N_2)和急性低氧(15.0% O_2,85.0% N_2)通气的状态下进行实验,监测各组大鼠心率(heart rate,HR)、左心室收缩压(left ventricular systolic pressure,LVSP)、右心室收缩压(right ventricular systolic pressure,RVSP)及左、右心室内压最大上升和下降速率(±dp/dt_(max))等心功能指标变化;同时,比较低氧通气前后动脉血气的变化。结果:常氧下,propranolol组、atenolol组与ICI 118,551组LVSP和左心室±dp/dt_(max)较给药前降低,同时propranolol组与atenolol组RVSP和右心室±dp/dt_(max)较给药前明显降低(P0.05)。低氧通气5 min后,各组大鼠与常氧组相比动脉血氧分压(PaO_2)、LVSP和左心室±dp/dt_(max)均降低(P0.05);但右心室±dp/dt_(max)明显升高(P0.05);且低氧条件下control组心功能指标的变化程度均比propranolol组和atenolol组明显。结论:低氧应激时心脏β_1-AR的激活可能是心脏发挥代偿调节的重要方式,但右心室通过紧张源性扩张代偿表现出的右心舒缩功能增强对低氧下机体循环血流量的维持更为重要。     

Involvement of the interleukin-2 receptor γ subunit in interleukin-4-dependent activation of mouse hematopoietic cells and splenic B cells

Interleukin-4 (IL-4) has various activities on B cells and on hematopoietic cells. We previously reported that TUGm2, a monoclonal antibody to the γ subunit of the IL-2 receptor (IL-2Rγ), inhibited IL-4-dependent proliferation of CTLL2, a cytotoxic T cell line. We proposed that IL-2Rγ is required for the functional IL-4 receptor (IL-4R) in T cells. In the present work, we further examined whether or not IL-2Rγ is involved in IL-4R function in mouse myeloid cell lines and splenic B cells. TUGm2 suppressed the IL-4-induced proliferation of BA/F3 or IC2 cells, as well as of purified splenic B cells. TUGm2 partially suppressed proliferation of B cells induced by the combination of IL-4 and anti-immunoglobulin M (IgM) antibody. In contrast, TUGm2 had no effect on proliferation of B cells induced by anti-IgM antibody alone or lipopolysaccharide (LPS). TUGm2 also inhibited IgE production induced by IL-4 of LPS-stimulated B cells. The induction of major histocompatibility complex class II molecules or CD23 by IL-4 was virtually unaffected by TUGm2 antibody. These results indicate that IL-2Rγ is differentially involved in various IL-4-dependent reactions.     

The role of alpha7 and alpha4beta2 nicotinic receptors in the nicotine-induced anxiolytic effect in zebrafish

Zebrafish (Danio rerio) have been widely used to study the molecular mechanisms of development including neurodevelopment. More recently, they have begun to be used to study neuropharmacology and neurotoxicology. Critical for this line of research are methods to study behavioral function in zebrafish. Previous studies have compared zebrafish with mammalian models to determine similarities and differences in locomotor behavior, learning and memory. Relatively little research has been conducted on stress response and anxiety behavior as well as the pharmacologic response in zebrafish. We have developed a test for zebrafish to assess stress response and anxiety: the novel tank diving test. In this short test normally zebrafish dive to the bottom of a novel tank and then gradually over the 5-min test begin exploring higher levels of the tank. Nicotine, which has anxiolytic effects in rodents and humans was found to diminish this novel tank diving response in zebrafish. The current study examined the nicotinic receptor subtype selectivity involved in the actions of nicotine. Two nicotinic receptor subtype selective antagonists were used: MLA (an α7 antagonist) and DHβE (an α4β2 antagonist). We replicated our previous finding of the anxiolytic effect of nicotine with significantly less bottom dwelling by the fish after nicotine treatment. This nicotine-induced anxiolytic effect was reversed by both MLA and DHβE, indicating that both nicotinic α7 and α4β2 receptors are involved in the nicotinic effect on anxiety in zebrafish.     

Markers in the 15q24 nicotinic receptor subunit gene cluster (CHRNA5‐A3‐B4) predict severity of nicotine addiction and response to smoking cessation therapy

Stopping smoking is difficult even with treatment. Many patients prescribed pharmacologic treatments for smoking cessation experience side effects or lack of efficacy. We performed a pharmacogenetic study of the efficacy and tolerability of bupropion and transdermal nicotine (TN), two treatments for smoking cessation. Samples were drawn from two studies. In the first study (Maintenance 1, MT1), 301 smokers received bupropion plus TN for 11 weeks, followed by 14 weeks of placebo or bupropion. In the second study (MT2), 276 smokers received bupropion and TN for 8 weeks. We focused on eight SNPs in the 15q24 region, which contains the genes for the nicotinic cholinergic receptor subunits CHRNA5, CHRNA3, and CHRNB4, and has previously been implicated in nicotine addiction and smoking cessation. Analyses of baseline smoking quantity (SQ) identified an association between SQ and both the functional CHRNA5 SNP rs16969968 (D398N) and the CHRNA3 SNP rs1051730 (Y215Y) in a combined cohort containing MT1 and MT2. An association between SQ and ethnicity was also identified in the combined cohort. Pharmacogenetic analysis showed a significant association between rs8192475 (R37H) in CHRNA3 and both higher craving after quitting and increased withdrawal symptoms over time in MT2. Two markers for point prevalence abstinence, CHRNA5 SNP rs680244 and CHRNB4 SNP rs12914008, were also identified in MT2, with the strongest findings at week 52. These results provide further support for the role of the CHRNA5/A3/B4 subunits in determining number of cigarettes smoked and response to smoking cessation therapy. © 2011 Wiley‐Liss, Inc.     

Lack of involvement of μ1 opioid receptors in dermorphin-induced inhibition of hypoxic and hypercapnic ventilation in rat pups

The effects of dermorphin, a mu-selective opioid agonist, on respiratory responses to altered O(2) and CO(2) during postnatal development were investigated in conscious, unrestrained Wistar rats aged 2-21 days. Respiration was recorded by barometric plethysmography. Dermorphin (4 mg kg(-1)) was administered subcutaneously, and the ventilatory responses to hypoxia (11% O(2), 89% N(2)) in 2-21-day-old pups and hyperoxia (100% O(2)), and hypercapnia (8% CO(2), 92% O(2)) in 2-13-day-old pups were assessed in the presence and absence of the mu(1) receptor antagonist naloxonazine (10 mg kg(-1) s.c.) administered 1 day before testing. Six minutes of hypoxia increased ventilation in all age groups, largely via an increase in frequency. Dermorphin inhibited the ventilatory response to hypoxia, and this inhibition was insensitive to naloxonazine. After 5 min of hyperoxia, ventilation was the same as with air breathing except in the presence of dermorphin, when hyperoxic ventilation was depressed by a naloxonazine-insensitive decrease in frequency. Following this 5 min 100% O(2) exposure, pups were exposed to hypercapnia, and respiratory parameters were measured 5 min later. The ventilatory response to CO(2) was inhibited by dermorphin in a naloxonazine-insensitive manner. There was no evidence for endogenous mu(1) receptor modulation of the ventilatory responses to altered gases in rat pups of any age. Thus, mu opioid-induced inhibition of the hypoxic and hypercapnic responses in young rats does not occur via activation of mu(1) opioid receptors.     

Characterization of β β β-ADRENERGIC receptors in fish and amphibian lymphoid organs

Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity β-adrenergic receptors specific for β-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian β¹ and β²-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another β-AR subtype, or a serotonergic receptor.