神经元烟碱受体研究进展

神经元烟碱受体 (nAChR)是由乙酰胆碱控制开放的阳离子通道。该受体由多种亚基组成 ,结构复杂 ,因而具有不同的药理学和生物物理学性质。本文根据有关nAChR的文献 ,从不同方面综述其研究进展     

神经元烟碱受体β2亚基基因克隆和原核细胞表达

采用异硫氰酸胍－酚－氯仿抽提法从孵育至１２天的鸡胚视叶顶盖中提取ＲＮＡ。ｃｈｏｅｐｆｅｒ报道的序列设计引物，引物的５端添加ＥｃｏＲＩ和ＸｂａＩ酶切位点。通过逆转录聚合酶链反应（ＲＴ－ＰＣＲ）得到１．４８ｋｂ长的ＣＤＮＡ片段，用ＥｃｏＲＩ和ＸｂａＩ酶切ＣＤＮＡ后插入质粒ＰＵＣ１９。测序采用Ｓａｎｇｅｒ双氧终止法，结果与文献报道一致。将β基因克隆入ＰＢＶ２２０原核表达载体中，宿主菌经４２℃诱导，经ＳＤ     

钾通道阻断剂四乙铵对卵巢癌细胞增殖及凋亡的影响

目的 研究钾通道阻断剂四乙铵(TEA)对卵巢癌细胞(SKOV3)凋亡的影响.方法 将浓度为2×104/ml的卵巢癌细胞分别设对照组和不同浓度(1、5、10、20mmol/L)TEA组.将TEA作用于卵巢癌细胞,用MTT法检测细胞活性,采用Hoechest33258染色检测细胞凋亡,同时通过碘化丙啶(PI)染色,采用流式细胞仪检测细胞凋亡比率.结果 在TEA浓度为1、5、10、20mmol/L时,对SKOV3细胞增殖的抑制率分别为8.61%、18.86%、46.63%和65.22%,表明TEA对SKOV3细胞的增殖有抑制作用并呈现明显的剂量依赖性,当TEA浓度=5mmol/L时,对SKOV3细胞增殖的抑制效应明显;Hoechst33258染色结果显示,TEA能诱导SKOV3细胞凋亡;流式细胞仪检测结果表明,SKOV3细胞经TEA浓度为1、5、10、20mmol/L处理后48h,其细胞凋亡率分别为9.23%、21.57%、54.22%和71.06%,与对照组(2.08%)比较,差异有显著性(P＜0.01),提示TEA可诱导SKOV3细胞凋亡,且TEA促进SKOV3细胞凋亡作用具有一定的剂量依赖性.结论 钾通道对SKOV3细胞增殖具有重要调控作用,钾通道阻断剂TEA阻断钾通道后可促进细胞凋亡.     

依色林阻断神经元烟碱受体作用机制的膜片钳研究

依色林阻断神经元烟碱受体作用机制的膜片钳研究郑建全何湘平杨爱珍刘传缋军事医学科学院毒物药物研究所北京１００８５０除了可逆性抑制胆碱酯酶的作用外，依色林尚可直接与烟碱受体相互作用，产生复杂的生物学效应，如对烟碱受体具有弱的激动作用；抑制肌肉终板电流并加...     

中枢烟碱受体β2亚基的基因克隆

中枢烟碱受体β２亚基的基因克隆李前洪孝庄军事医学科学院毒物药物研究所北京１００８５０国内中枢烟碱受体的分子生物学研究起步较晚，目前水平与国外差距较大。我们所进行的研究工作，是为今后烟碱受体的分子药理学研究以及受体药物过筛模型的建立，创造条件。中枢烟碱...     

吸烟对外周血单个核细胞表达烟碱乙酰胆碱受体的影响

目的研究健康男性外周血单个核细胞（PBMCs）烟碱型乙酰胆碱受体（nAchR）亚型的表达情况及吸烟对其表达量的影响。方法抽取92例健康男性吸烟者和36例不吸烟者的外周静脉血,通过半定量RT-PCR对其nAchR11种亚基（α2～α7、α9、α10、β2～β4）mRNA在PBMCs的中表达情况进行分析。结果11种nAchR亚基只有α2、α5、α6、α7四种亚基mRNA在吸烟者和不吸烟者PBMCs中有表达,吸烟者α5、α7亚基mRNA的表达量比不吸烟者低（均P〈0.01）,而两者α2亚基mRNA的表达无显著性差异（P〉0．05）。结论烟碱与外周血免疫细胞上nAchR的相互作用可能是吸烟导致机体免疫系统功能改变的因素之一。     

烟碱对大鼠脑突触体摄取钙离子的影响

在大鼠脑突触体上，０．０１～１００μｍｏｌ／Ｌ的Ｎ受体激动剂烟碱可浓度依赖性地增加对同浓度４５Ｃａ２＋（１．０μｍｏｌ／Ｌ）的摄取量，并为１．０μｍｏｌ／ＬＮ受体拮抗剂美加明所对抗。相同条件下，１．０μｍｏｌ／Ｌ烟碱也可显著增加脑突触体对不同浓度４５Ｃａ２＋（０．１～１００μｍｏｌ／Ｌ）的摄取量。提示烟碱作用于脑Ｎ受体，可增加脑突触体对钙离子的摄取量     

槟榔碱对大鼠脑肌醇含量的影响以及与烟碱的相互作用

急性实验中，经Ｍ受体激动剂槟榔碱５０，１００，２００ｍｇ／ｋｇ（ｉｐ）３０ｍｉｎ后，在１００和２００ｍｇ／ｋｇ剂量下可显著地提高大鼠大脑皮层和海马中肌醇含量，但不影响纹状体中肌醇含量；Ｍ受体拮抗剂阿托品２ｍｇ／ｋｇ可对抗槟榔碱２００ｍｇ／ｋｇ（ｉｐ）提高大脑皮层和海马中肌醇含量的作用；反复注射Ｎ受体激动剂烟碱０．５，１，１，２，２ｍｇ／ｋｇ（ｉｐ）与槟榔碱５０ｍｇ／ｋｇ（ｉｐ）之间存在协同作用，进一步提高大脑皮层和海马中肌醇含量。慢性实验中，给槟榔碱２，５，１０ｍｇ／ｋｇ（ｓｃ）每日２次１４ｄ后，在１０ｍｇ／ｋｇ剂量下可显著地提高大脑皮层中肌醇含量；阿托品２ｍｇ／ｋｇ对抗槟榔碱１０ｍｇ／ｋｇ的作用；烟碱２ｍｇ／ｋｇ与槟榔碱５ｍｇ／ｋｇ之间存在协同作用，进一步提高大脑皮层中肌醇含量，烟碱的这一作用可为美加明１ｍｇ／ｋｇ所对抗。提示槟榔碱激动中枢Ｍ受体提高大脑皮层和海马中肌醇含量，反复注射烟碱可提高Ｍ受体对其激动剂的敏感性     

碘化二甲基粉防己碱对骨骼肌细胞烟碱诱发电流的抑制作用

目的：比较碘化二甲基粉防己碱（DMTI）和二氢－β－刺桐啶碱（dHβE)对烟碱诱发电流的抑制作用，并确定DMTI在烟碱受体上的作用位点，方法：取新生Wistar大鼠骨骼肌细胞体外培养获得肌球，用膜片箝全细胞记录技术，观察肌球的烟碱诱发电流。结果：0．08mmol.L^-1 DMTI抑制烟碱诱发电流幅度的作用与0．02mmol.L^-1dHβE的作用类似，向单个细胞持续给烟碱覆盖诱发电流的全过程，电流的衰减过程反映了烟碱受体的失敏，DMTI使烟碱诱发电流的慢失敏时间常数减小，即加速受体失敏，但没发现dHβE有这种作用，在－30，－50，－70，－90mV钳制电压处观察电压改变对DMTI和对DHβE作用的影响，没有发现DMTI或dHβE抑制烟碱电流的作用随钳制电压改变而改变，即两的作用都是非电压依赖性的，表明DMTI和dHβE都不是离子通道阻断剂。结论：由于烟碱受体是变构蛋白，变构调节剂能加速受体失敏，DMTI的抑制作用符合变构调节剂的作用特点，并且作用位点不在烟碱受体离子通道，而dHβE不是变构调节剂。     

突触前状态对梭曼阻断神经节作用的影响——Ⅱ.刺激节前神经对梭曼阻断上颈交感神经节的影响

用空气间隙及蔗糖间隙法分别记录大鼠及兔离体上颈交感神经节电位的慢变化,在不刺激节前神经的大鼠、兔实验中,梭曼不引起节电位去极化。在刺激节前交感神经干的状态下,梭曼引起神经节去极化,去极化电位持续整个实验过程。梭曼引起的去极化电位可为HI-6所恢复,但不被六甲溴铵、东莨菪硷所影响,也不因停止刺激节前神经而自然恢复。本文对刺激节前在梭曼阻断神经节作用中的地位进行了讨论。     

[I] 5-Iodo-3-pyridyl ethers: syntheses and binding to neuronal nicotinic acetylcholine receptors

Three 3-pyridyl ether nicotinic ligands-(S)-5-Iodo-3-[(2-pyrrolidinyl)-methoxy]pyridine (5-iodo-A-85865), (S)-5-Iodo-3-[1-(methyl)-2-pyrrolidinyl-methoxy]pyridine (5-Iodo-A-84543), and (S)-5-iodo-3-[1-methyl-(2-azetidinyl)-methoxy]pyridine (5-iodo-N-Me-A-85380) were labeled with I-125/I-123, and their ability to label high-affinity brain nicotinic acetylcholine receptors (nAChRs) was evaluated. The most promising ligand, [^123/125I] 5-iodo-A-85865, showed approximately 65% inhibition of radioactivity uptake in thalamus in mice pretreated with cytisine. Preliminary SPECT imaging studies with [¹²³I] 5-iodo-A-85865 revealed a distribution profile consistent with nAChRs (thalamus > frontal cortex > cerebellum) and a more rapid pharmacokinetic profile relative to azetidinyl 3-pyridyl ether based ligands.     

Quantification of nicotinic acetylcholine receptors in human brain using [123I]5-I-A-85380 SPET

The purpose of this study was to assess the utility of a new single-photon emission tomography ligand, [¹²³I]5-iodo-3-[2(S)-2-azetidinylmethoxy]pyridine (5-I-A-85380), to measure regional nAChR binding in human brain. Six healthy nonsmoker subjects (two men and four women, age 33±15 years) participated in both a bolus (dose: 317±42 MBq) and a bolus plus constant infusion (dose of bolus: 98±32 MBq, B/I=6.7±2.6 h, total dose: 331±55 MBq) study. The study duration was 5–8 h and 14 h in the former and the latter, respectively. Nonlinear least-squares compartmental analysis was applied to bolus studies to calculate total (V_T) and specific (V_S) distribution volumes. A two-tissue compartment model was applied to identify V_S. V_T was also calculated in B/I studies. In bolus studies, V_T was well identified by both one- and two-tissue compartment models, with a coefficient of variation of less than 5% in most regions. The two-compartment model gave V_T values of 51, 22, 27, 32, 20, 19, 20, and 17 ml cm^–3 in thalamus, cerebellum, putamen, pons, and frontal, parietal, temporal, and occipital cortices, respectively. The two-compartment model did not identify V_S well. B/I studies provided poor accuracy of V_T measurement, possibly due to deviations from equilibrium conditions. These results demonstrate the feasibility of quantifying high-affinity type nAChRs using [¹²³I]5-I-A-85380 in humans and support the use of V_T measured by bolus studies.     

Radiosynthesis and ex vivo evaluation of [11C]-SIB-1553A as a PET radiotracer for beta4 selective subtype nicotinic acetylcholine receptor

[¹¹C]-SIB-1553A ((±)-4-[2-((N-[¹¹C]-methyl)-2-pyrrolidinyl)ethyl]thiophenol) was labelled with carbon-11 (t_1/2=20.4 min) and evaluated in vivo as potential radiotracer for noninvasive assessment of the β4 subunit nicotinic acetylcholine neurotransmission system with positron emission tomography (PET). The labelling precursor was obtained within five steps from N-Boc-prolinal in 45–56% overall yields. The radiosynthesis of [¹¹C]-SIB-1553A was achieved by a selective N-[¹¹C]-methylation in 32 min with a radiochemical purity greater than 97%, 7.5–30 GBq/μmol of specific radioactivity and 55–65% radiochemical yield (decay corrected, based on [¹¹C]methyl iodide). The ex vivo pharmacological profile of [¹¹C]-SIB-1553A was evaluated in rats with biodistribution studies in organs and in brain structures by autoradiography. The radiotracer uptake in the brain reached 0.49 %ID/g at 10 min and no brain radiometabolite was detected 40 min after intravenous injection. The quantification of radioactivity in various cerebral structures indicated a significantly higher radioactivity level at 15 min than at 30 min. Among the β4 nAChR subunit-rich structures studied in the rat brain, only the thalamus at 15 and 30 min and the hippocampus at 30 min showed significantly higher uptake. Moreover, competition studies performed with SIB-1553A (15 min before the radiotracer injection) revealed only a low specific binding estimated to 7% of the total binding at 15 min and 13% at 30 min.     

牛m1和m5受体内环3区的克隆及主动脉内皮细胞上两种受体mRNA表达的检测

目的 :克隆牛m1和m5受体亚型特异性区域内环 (innerloop) 3区 ,并检测牛主动脉内皮细胞上两种受体mRNA的表达。方法 :根据人、小鼠的m1和m5受体序列 ,设计并合成针对两种受体内环 3区的特异性引物。采用RT_PCR法从牛海马组织中扩增牛m1和m5受体内环 3区 ,并通过半定量RT_PCR法检测两种受体mRNA在传代培养前后的牛主动脉内皮细胞中的表达。结果 :序列测定与对比表明克隆得到了牛m1和m5受体内环 3区。半定量RT_PCR结果显示 ,原代牛主动脉内皮细胞表达m1受体mRNA而未检测到m5受体mRNA ,两种受体mRNA在传 10代的培养牛主动脉内皮细胞中均未检测到有表达。结论 :本研究为探讨血管内皮细胞上介导乙酰胆碱诱发血管舒张反应的受体与M受体的关系提供了线索     

Evaluation of radioiodinated 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine as a ligand for SPECT investigations of brain nicotinic acetylcholine receptors

5-Iodo-3-(2(S)-azetidinylmethoxy)pyridine (5IA), an A-85380 analog iodinated at the 5-position of the pyridine ring, was evaluated as a radiopharmaceutical for investigating brain nicotinic acethylcholine receptors (nAChRs) by single photon emission computed tomography (SPECT). [123/125I]5IA was synthesized by the iododestannylation reaction under no-carrier-added conditions and purified by high-performance liquid chromatography (HPLC) with high radiochemical yield (50%), high radiochemical purity (> 98%), and high specific radioactivity (> 55 GBq/micromol). The binding affinity of 5IA for brain nAChRs was measured in terms of displacement of [3H]cytisine and [125I]5IA from binding sites in rat cortical membranes. The binding data revealed that the affinity of 5IA was the same as that of A-85380 and more than seven fold higher than that of (-)-nicotine, and that 5IA bound selectively to the alpha4beta2 nAChR subtype. Biodistribution studies in rats indicated that the brain uptake of [125I]51A was rapid and profound. Regional cerebral distribution studies in rats demonstrated that the accumulation of [125I]5IA was consistent with the density of high affinity nAChRs with highest uptake observed in the nAChR-rich thalamus, moderate uptake in the cortex and lowest uptake in the cerebellum. Administration of the nAChR agonists (-)-cytisine and (-)-nicotine reduced the uptake of [125I]5IA in all regions studied with most pronounced reduction in the thalamus, and resulted in similar levels of radioactivity throughout the brain. [125I]5IA binding sites were shown to be saturable with unlabeled 5IA. Behavioral studies in mice demonstrated that 5IA did not show signs of behavioral toxicity. Furthermore, SPECT studies with [123I]5IA in the common marmoset demonstrated appropriate brain uptake and regional localization for a high-affinity nAChR imaging radiopharmaceutical. These results suggested that [123I]5IA is a promising radiopharmaceutical for SPECT studies of central nAChRs in human subjects.     

雌激素对穹隆海马伞切断大鼠脑内nAchR的干预作用

目的以双侧穹隆-海马伞切断制作阿尔茨海默病(AD)大鼠模型,观察脑内海马CA1区和皮层区烟碱型乙酰胆碱受体(nAchR)表达的变化,探讨与AD相关的发病机制,并观察雌激素的干预作用。方法健康雌性W istar大鼠,随机分为3组:(1)穹隆-海马伞切断组;(2)雌激素治疗组;(3)假手术对照组;每组大鼠5只。在脑立体定位仪上切断穹隆-海马伞,建立模拟AD动物模型。应用免疫组织化学技术观察AD大鼠海马CA1区和皮层区的nAchR阳性细胞表达的变化,以及应用外源性雌激素的干预作用。结果与对照组大鼠脑内nAchR表达比较,穹隆-海马伞切断组大鼠脑内海马CA1区、皮层区nAchR表达显著减少(P<0.05);与穹隆-海马伞切断组比较,雌激素治疗组海马CA1区、皮层区nAchR表达显著增加(P<0.01),但与对照组比较无显著差异(P>0.05)。结论穹隆-海马伞切断组大鼠脑内海马CA1区、皮层区nAchR的阳性细胞显著减少,补充外源性雌激素治疗后,上述各脑区内的nAchR表达显著增加。nAchR的变化与AD的病理变化过程密切相关,补充雌激素可以干预这种病理过程。     

交感神经系统在辐射免疫效应中的调节作用

大鼠头部受10Gyx射线照射后48小时, 脾脏中NE和E含量明显增高, 同时伴有脾细胞对PWM的反应性下降和对ConA的反应性增强, 脾与胸腺重量减轻, 外周血中有核细胞数增加。当大鼠经利血平处理后再进行头部照射, 上述免疫学参数的变化基本消失。此外, 大鼠头部照射后立即用SRBC免疫, 4天后其脾细胞抗体形成能力明显增强, 而在24小时后免疫动物, 则无明显改变。这些结果表明, 头部照射所引起的大鼠免疫功能的变化与交感神经系统的兴奋性增强确密切关系。     

嗅球成鞘细胞对新生大鼠皮层神经元的影响

目的　观察嗅球成鞘细胞 (OECs)培养液对培养中的新生大鼠大脑皮层神经元的影响。方法　原代培养新生大鼠OECs和大脑皮层神经元 ;收集含有OECs分泌物的培养液并以不同剂量添加到正在培养的神经元中 ,观察神经元正常及损伤时的生长情况。结果　培养的OECs形态学观察及神经生长因子受体 (p75NGFR)、胶质纤维酸性蛋白 (GFAP)、胶质细胞源性营养因子 (GDNF)免疫组化阳性反应均与报道一致。OECs培养液 (0 .4～ 1.0ml)能明显促进神经元生长速度 ,并能有效地减少受损神经元的崩解。结论　OECs有促进正常神经元生长、保护损伤神经元的作用 ,这可能与OECs分泌多种生物活性物质有关     

5-[<Superscript>123</Superscript>I]Iodo-A-85380: assessment of pharmacological safety,radiation dosimetry and SPECT imaging of brain nicotinic receptors in healthy human subjects

Recently, 5-[123I]iodo-3-(2(S)-azetidinylmethoxy)pyridine ([123I]5IA) was developed as a ligand for imaging the nicotinic acetylcholine receptor (nAChR) in human brain using single photon emission computed tomography (SPECT). In the present study, the toxicity and radiation absorbed dose of [123I]5IA were investigated. Behavior and physiological parameters were examined in mice and rats after administration of 5IA. There were no changes in these parameters in animals administered 1 microg/kg of 5IA or less, indicating that the no observed effect level (NOEL) of 5IA was 1 microg/kg. [123I]5IA was then administered to healthy human subjects and serial whole-body images were acquired over 24 hr. Initially, high levels of radioactivity were observed in the liver and urinary bladder and moderate levels in the lungs, kidneys, and brain. Whole brain activity at 1 hr was 4.6 +/- 0.4% of the injected dose and this value gradually decreased with time. The majority (-75%) of the radioactivity was excreted in urine within 24 hr, and less than 1% remained in all organs tested. The biological half-life of [1231]51A averaged 7.2 +/- 4.0 hr. Based on the biodistribution data, radiation absorbed doses were estimated using MIRDOSE 3.1 software with the dynamic bladder model and the ICRP gastrointestinal (GI) tract model. Consequently, the effective dose equivalent was estimated to be 30 +/- 1.4 microSv/MBq, which is an acceptable radiation burden. Having determined the safety of this compound, we performed SPECT imaging in a healthy human subject using 171 MBq of [123I]5IA. SPECT images clearly revealed a cerebral distribution of radioactivity that was consistent with the known distribution of central nAChRs in humans. These results suggest that [123I]5IA is a promising ligand for imaging nAChRs in humans, with an acceptable dosimetry and pharmacological safety at the dose required for adequate SPECT imaging.