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.

Effect of green flickering light on myopia development and expression of M1 muscarinic acetylcholine receptor in guinea pigs

AIM： To investigate the effects of green flickering light on refractive development and expression of muscarinic acetylcholine receptor（mAChR） M1 in the eyes of guinea pigs.METHODS： Thirty guinea pigs（15-20 days old） were randomly divided into three groups（n=10/group）. Animals in group I were raised in a completely closed carton with green flickering light illumination. Those in group II were kept in the open top closed carton under normal natural light. Guinea pigs were raised in a sight-widen cage under normal natural light in group III. The refractive status and axial length were measured before and after 8 weeks＇ illumination. Moreover, total RNA extracted from retinal, choroidal, and scleral tissues were determined by real-time reverse transcription polymerase chain reaction（RT-PCR）. The expressions of the receptor M1 were also explored in the retina, choroid, and sclera using immunohistochemistry.RESULTS： There was a remarkable reduction in refractive error and increase in axial length after 8-weeks＇ green flickering light stimulation（P〈0.001）. The expression of M1 receptor mRNA in sclera and retina in myopia group were remarkably lower than that in group II and III（P〈0.01）. Significant reduced expression of M1 receptor stimulated by green flickering light in retina and sclera tissues were also observed（P〈0.05）. However, there was no M1 receptor expression in choroid in 3 groups.CONCLUSION： Myopia can be induced by 8 weeks＇ green flickering light exposure in the animal model. M1 receptor may be involved causally or protectively in myopia development.

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.

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.

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.

M_(4) muscarinic receptors regulates dopamine/DARPP-32 signaling and glutamate transmis⁃sion to balance dopaminergic D1 function in mouse dorsal striatum

OBJECTIVE Abnormal striatal dopaminergic and glutamatergic neurotransmis⁃sion is central to the pathophysiology of schizo⁃phrenia.In this study,we investigated the roles of M4 receptor interplay with D1 signaling in stria⁃tal neurotransmission that affect glutamatergic transmission to control the etiology of neuropsy⁃chiatric disorders.METHODS To study dorsal striatum(DS)region-specific neuronal and behav⁃ioral responses modulated by M4 receptors,we used clustered regularly interspaced short palin⁃dromic repeats-associated protein 9 technology to generate mice lacking M4 in the dorsal stria⁃tum(DS-M4-KD).The M4 positive allosteric modu⁃lator,VU0467154,were used to study the phar⁃macologically profiles with M4 receptor stimula⁃tion in WT mice.Oxotremorine M(Oxo-M),a no subtype-selective muscarinic agonist,was used to show that mAchRs activation,in order to dissect the particular function of M4,in DS-M4-KD mice.Open filed test and forced swim test were used to assess the change of psychiatric-like behav⁃iors.Western blotting and immunohistochemistry were used to detect protein levels of phosphory⁃lation site of dopamine-and cAMP-regulated phosphoprotein of 32 ku(DARPP-32).Whole-cell patch-clamp recording was used to assess M4-mediated cholinergic inhibition of glutamater⁃gic synaptic input transmission.RESULTS West⁃ern blotting and immunohistochemistry assay showed VU0467154(5 mg·kg-1,ip)promoted phosphorylation of DARPP-32 at Thr75,and atten⁃uated D1-dependent phosphorylation of DARPP-32 at Thr34 within the mouse DS.Consistently,the Oxo-M(4μg,icv)also increased DARPP-32 phosphorylation at site Thr75 to reversed phos⁃phorylation at site Thr34 in WT mice,but not in DS-M4-KD mice.In parallel with altered DARPP-32 responses,VU0467154 or Oxo-M evoked a psychological stress response and reversed D1-induced hyperlocomotion in mice in open field test and force swim tests.However,Oxo-M sup⁃pression of D1-depengdeng behavioral respons⁃es was impaired in DS-M4-KD mice.Whole-cell patch recording showed that VU0467154 or Oxo-M mediated endogenous cholinergic inhibition of miniature excitatory postsynaptic currents through M4 receptors,which in turn suppressed D1-depen⁃dent glutamatergic synaptic transmission in the DS.CONCLUSION This study provides evidence for the role of M4 receptors in regulation of dopa⁃mine/DARPP-32 signaling and glutamate respons⁃es in the DS,and therefore modulation of psychi⁃atric behaviors associated with D1 signaling.This results indicate the mechanisms of treatments targeting M4 in psychiatric disorders.

Preparation of an imaging agent for cerebral muscarinic acetylcholine receptor, (R,S)(131)~I-QNB

The method to synthesize a high affinity muscarinic receptor antagonist (R,S)I-QNB[(R)-(-)-1-azabicyclo [2,2,2]oct-3-yl-(S)-(+)-α-hydroxy-α-(4-[127I]iodophenyl)-α-phenyl acetate] from 4-nitrobenzophenone with improvement compared to literatures was reported in this article. IR, MS and 1HNMR characterized the final product. (R,S)131I-QNB was prepared using Cu(I) assisted iodine exchange labeling, and showed by TLC that the radiolabeling yield(RLY) was over 80%, and radiochemical purity(RCP) was over 95%. Stability of the labeled compound was also determined. It was found that (R,S)131I-QNB dried by nitrogen blowing can stay at 4-10℃ for a week without change of RCP.

电针肾俞穴对肾阳虚近视豚鼠视网膜M1受体表达的影响

目的:观察电针肾俞穴对肾阳虚近视豚鼠生长发育、视力及视网膜毒蕈碱样乙酰胆碱(mAChR)M1受体表达的影响,为肾阳虚近视的治疗提供依据。方法:将60只豚鼠随机分为正常对照(NC)组、透镜诱导近视(LIM)组、肾阳虚近视假穴(KYDM+SHAM)组、肾阳虚近视电针(KYDM+EA)组,每组15只。NC组不采取干预措施;LIM组豚鼠右眼配戴-6.00 D透镜构建近视模型;其余两组在戴镜基础上连续2周腹腔注射氢化可的松(剂量10 mg/kg)构建肾阳虚近视模型,造模2周后,KYDM+EA组电针豚鼠双侧肾俞穴,KYDM+SHAM组电针豚鼠臀部假穴,共4周。观察造模不同时间各组豚鼠一般情况,测量体质量和肛温,检测双眼屈光度和眼轴长度,检测血清游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)、睾酮(T)、雌二醇(E_(2)),并在造模结束后采用定量聚合酶链反应(Q-PCR)、免疫组织化学和蛋白质印迹法检测视网膜M1受体mRNA和蛋白表达水平。结果:造模2周后,KYDM+SHAM组和KYDM+EA组豚鼠出现毛发枯槁、无光泽及消瘦、拱背眯眼、倦怠懒动、畏寒肢冷等肾阳虚症状,血清FT3、FT4、T水平低于LIM组,E_(2)高于LIM组,体质量、肛温以及右眼屈光度均低于LIM组,眼轴长度长于LIM组,差异均有统计学意义(P<0.05)。电针4周后,KYDM+EA组豚鼠肾阳虚症状有效改善,血清激素水平趋于正常,与KYDM+SHAM组相比,体质量、肛温、右眼屈光度均明显升高,右眼眼轴长度缩短,视网膜M1受体mRNA和蛋白表达水平降低,差异均有统计学意义(P<0.05)。与LIM组相比,KYDM+EA组豚鼠体质量增加,差异有统计学意义(P<0.05);肛温、右眼屈光度和眼轴长度、视网膜M1受体mRNA和蛋白表达水平差异均无统计学意义(P>0.05)。结论:电针肾俞穴有利于促进肾阳虚近视豚鼠生长发育恢复及视力改善,机制可能与降低视网膜中M1受体表达有关。

高原低氧暴露下海马中M1AChR表达对雄性大鼠学习和记忆的影响

目的 考察高原低氧暴露下海马中毒蕈碱型乙酰胆碱受体M1(muscarinic acetylcholine receptor M1,M1 AChR)表达对雄性大鼠学习和记忆的影响。方法 将大鼠随机分为常氧对照组(NC组)、常氧组(N组)、低氧组(H组)和低氧+TAK-071组(HT组)。采用定位巡航实验和空间探索实验分别考察大鼠的学习和空间记忆能力;采用苏木素-伊红染色(hematoxylin-eosin staining,H&E)技术观察大鼠额叶和海马的组织形态;采用免疫组织化学(immunohistochemical,IHC)染色和蛋白质印迹(Western Blot,WB)技术测定大鼠海马组织中M1 AchR的蛋白表达水平;采用荧光定量PCR(quantitative PCR,qPCR)技术测定大鼠海马组织中M1 AchR和叉头框蛋白P2(forkhead box protein P2,FOXP2)的基因表达水平。结果 经过Morris水迷宫(morris water maze,MWM)训练后,大鼠海马组织中M1 AChR的蛋白总体表达水平明显降低(P<0.05),但在海马CA1区、CA3区显著增加(P<0.05)。低氧环境会明显改变大鼠在定位巡航实验中的表现:逃避潜伏期时长增加;明显改变大鼠在空间探索实验中的表现:在目标象限的停留时间减少(P<0.05)。激活M1AChR后,大鼠海马组织中因低氧增加的M1 AChR明显降低(P<0.05)。结论 激活M1 AChR可以改善高原低氧对雄性大鼠的记忆损伤,减缓记忆衰退进程;对学习改善无影响。推测M1 AChR在雄性大鼠认知功能中的作用可能是保持已有记忆。

拟黑多刺蚁mAchR基因发育表达的荧光实时定量RT-PCR检测

【目的】研究毒蕈碱型乙酰胆碱受体(Muscarinic cholinergic receptor,mAchR)在拟黑多刺蚁个体发育中的表达情况,探讨其是否参与拟黑多刺蚁的个体发育行为。【方法】采用SYBRGreenⅠ实时定量RT-PCR方法,检测拟黑多刺蚁卵、幼虫、蛹和成虫个体发育阶段mAchR基因mRNA的相对表达量。【结果】mAchR基因在拟黑多刺蚁整个发育过程及各个品级成虫中均有表达。在卵期mAchR mRNA的表达量较幼虫期和蛹期高,而成虫期的表达量最高,可达卵期的3倍以上。3个成虫品级之间,工蚁的表达量最高,雄蚁和雌蚁的表达量在统计学上差异不显著。【结论】mAchR基因参与了拟黑多刺蚁的发育行为,而其主要功能可能是在调节成虫更为复杂的行为中起作用。

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination

During the development of the nervous system,there is an overproduction of neurons and synapses.Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening.We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway,at the neuromuscular junction,in the axonal development and synapse elimination process versus the synapse consolidation.The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination,in relation to other molecular pathways that we and others have found to regulate this process.In particular,we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors,coupled to downstream serine-threonine protein kinases A and C(PKA and PKC)and voltage-gated calcium channels,at different nerve endings in developmental competition.The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site,influence each other,and require careful studies to individualize the mechanisms of specific endings.We describe an activity-dependent balance(related to the extent of transmitter release)between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals.The downstream displacement of the PKA/PKC activity ratio to lower values,both in competing nerve terminals and at postsynaptic sites,plays a relevant role in controlling the elimination of supernumerary synapses.Finally,calcium entry through L-and P/Q-subtypes of voltage-gated calcium channels(both channels are present,together with the N-type channel in developing nerve terminals)contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination(the weakest in acetylcholine release and those that have already become silent).The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development.Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.

M3受体遗传变异与血压钠钾反应性的关联研究

目的M3毒蕈碱乙酰胆碱受体(M3受体)由CHRM3基因编码,广泛分布于心血管系统并发挥重要的心脏调节作用。本研究旨在探讨M3受体遗传变异与钠钾饮食干预后血压反应性之间的关系。方法从中国陕西省农村地区招募来自124个家庭的333名受试者,经过3 d的基线观察后,依次进行7 d低盐饮食、7 d高盐饮食、7 d高盐补钾饮食。选取13个CHRM3基因单核苷酸多态性(SNP)位点进行相关分析。结果CHRM3基因SNP位点rs10802811与低盐期及高盐期舒张压及平均动脉压反应性显著相关,而rs6429147、rs373288072、rs114677844、rs663148与高盐期收缩压及平均动脉压反应性显著相关。此外,rs6692904与高盐补钾期收缩压、舒张压及平均动脉压反应性显著相关。结论M3受体基因多态性与血压钠钾反应性相关,提示M3受体基因可能参与血压盐敏感性及钾敏感性的形成。

益气养阴祛瘀方对干燥综合征NOD小鼠颌下腺AQP5、M3R表达的影响

目的研究益气养阴祛瘀方(黄芪、生地黄、玄参、丹参、益母草等)对干燥综合征NOD小鼠颌下腺水通道蛋白5(AQP5)以及毒蕈碱样胆碱能受体3(M3R)表达的影响。方法将30只NOD小鼠随机分为5组:模型组、西药组(硫酸羟氯喹60 mg·kg^(-1))及益气养阴祛瘀方低、中、高剂量组(11.147、22.295、44.590 g·kg^(-1)),每组6只。每日灌胃给药1次,连续给药8周。给药后第0、4、8周连续测量小鼠每日饮水量;给药结束后,分离小鼠双侧颌下腺并称定其质量,计算小鼠颌下腺指数;采用HE染色法进行颌下腺组织病理学观察,计算灶性指数评分;RT-qPCR法检测小鼠颌下腺组织AQP5、M3R mRNA表达水平;Western Blot法检测小鼠颌下腺组织AQP5、M3R蛋白表达水平;免疫组化法检测颌下腺组织AQP5蛋白表达水平;ELISA法检测血清干扰素γ(IFN-γ)、白细胞介素10(IL-10)含量。结果给药前,与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的日饮水量无明显变化(P>0.05);给药第4、8周,与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的日饮水量均显著减少(P<0.01)。模型组小鼠颌下腺淋巴细胞浸润明显并形成较多浸润灶;与模型组比较,益气养阴祛瘀方低、中、高剂量组及西药组NOD小鼠的颌下腺淋巴细胞浸润情况得到明显改善,浸润灶数量明显减少,灶性指数评分显著降低(P<0.01),颌下腺组织AQP5、M3R蛋白表达水平明显升高(P<0.05,P<0.01),血清IL-10水平明显升高(P<0.05,P<0.01);益气养阴祛瘀方高剂量组和西药组NOD小鼠的颌下腺指数明显升高(P<0.05);益气养阴祛瘀方中、高剂量组和西药组NOD小鼠颌下腺组织AQP5、M3R mRNA表达明显上调(P<0.05,P<0.01),血清IFN-γ含量显著降低(P<0.01)。结论益气养阴祛瘀方对NOD小鼠唾液腺损伤具有改善作用,可能通过上调AQP5、M3R表达,抑制颌下腺淋巴细胞浸润,改善唾液分泌功能,从而减缓干燥综合征进程。

海马M_2受体调节杏仁核内谷氨酸的释放与学习记忆的研究

目的研究海马内M1和M2受体对学习记忆功能的影响及其可能机制。方法SD大鼠分为3组,分别海马内注射等体积的M1-ASODN(M1-AS组)、M2-ASODN(M2-AS组)和生理盐水(对照组),以被动逃避反应的步入潜伏期作为衡量学习记忆功能的指标,以CZE-LIFD法检测杏仁核内游离谷氨酸的浓度。结果M1-ASODN缩短步入潜伏期,为对照组的25%(P<0.01),M2-ASODN使步入潜伏期较对照组增加了75%(P<0.05)。双侧海马内注射ASODN后与对照组相比,M2-AS组杏仁核内谷氨酸的含量增加164%(P<0.01),而M1-AS组无变化(P>0.05)。结论海马内M受体参与恐惧性记忆的调节,M2受体抑制学习记忆,M1受体促进学习记忆。海马内M2受体可能通过调节杏仁核内谷氨酸含量的变化介导被动逃避反应的记忆过程。

大鼠心室肌M_3受体与间隙连接蛋白43作为抗心律失常药物靶点的综合研究

目的在蛋白质分子水平研究心律失常相关蛋白质M3受体与间隙连接蛋白43之间的结构相互作用,并为其作为筛选药物靶点提供依据.方法通过免疫组化结合激光共聚焦显微镜,及免疫沉淀与免疫印迹技术,研究M受体与间隙连接蛋白43的结构性共定位关系.结果证实了大鼠心室肌细胞膜蛋白中M1～M5等5个亚型的存在;观察到大鼠单个心肌细胞膜上M3受体与间隙连接蛋白43的结构性共定位;发现M受体各亚型与间隙连接蛋白43均存在结构整合,且一定浓度离子型去垢剂可破坏M3受体与间隙连接蛋白43的结构整合关系,并进一步发现参与M3受体结构整合的是间隙连接蛋白43的磷酸化形式.结论大鼠心室肌M受体亚型与间隙连接蛋白43的磷酸化形式存在结构性共定位关系,且可被一定浓度离子型去垢剂破坏.

盐酸小檗碱对毒蕈碱型受体的作用

目的：研究盐酸小檗碱对乙酰胆碱系统的影响。方法：采用离体血管环、离体支气管螺旋体及放射配基受体结合实验等方法。结果：较低浓度的盐酸小檗碱（Ｂｅｒ）对乙酰胆碱的舒张反应有浓度依赖性的增强作用；而较高浓度的Ｂｅｒ可浓度依赖性地直接舒张带内皮的动脉环，此作用在去内皮或阻断Ｍ受体后被完全抑制。在豚鼠离体气管条上，Ｂｅｒ可引起浓度依赖性的收缩反应，而阿托品可阻断这种作用。放射配基受体结合实验显示盐酸小檗碱可特异性与大鼠脑组织的毒蕈碱受体结合，Ｋｉ值为１６μｍｏｌ·Ｌ－１。结论：盐酸小檗碱可激动毒蕈碱受体，从而引起血管的内皮依赖性的舒张反应及支气管的收缩。

介导乙酰胆碱诱发内皮依赖性血管舒张反应受体的特性

目的 针对血管内皮细胞上是否存在M受体的疑点 ,从基因水平和信号转导机理上探讨介导乙酰胆碱 (ACh)诱发内皮依赖性血管舒张反应受体的特性。方法 以新鲜分离和传代培养的牛主动脉内皮细胞为实验材料 ,比较两种内皮细胞M1～M5受体mRNA的分布状况、ACh诱发细胞内游离钙离子浓度 ([Ca2 + ]i)、环腺苷酸 (cAMP)含量变化。①设计高选择性、特异性寡核苷酸探针 ,采用点杂交方法对内皮细胞M1～M5受体mRNA进行分析 ,并通过反转录 聚合酶链反应 (RT PCR)、DNA序列测定及同源性分析确证点杂交的实验结果 ;②以Fluo 3和Fura 2为荧光探针 ,用共聚焦和荧光光度仪测定内皮细胞 [Ca2 + ]i 变化 ;③内皮细胞cAMP含量采用12 5I放射免疫分析方法测定。结果 在新鲜分离的牛主动脉内皮细胞存在M1～M4 受体mRNA ,内皮细胞经传代培养后仅检测到M4 受体mRNA ;1和 10μmol·L- 1ACh能够引起原代培养内皮细胞 [Ca2 + ]i增加 ,最大增加量为 12和 19nmol·L- 1;ACh激活原代培养内皮细胞的钙离子信号 ,表现为单次或连续2次的钙振荡 ,具有异时性和快速耐受的特点 ;此外 ,随ACh浓度增加 ,原代培养内皮细胞cAMP含量均呈微弱增加趋势 ;ACh不引起传代培养内皮细胞[Ca2 + ]i 浓度的改变 ,但能浓度依赖地降低cAMP含量。结论 新鲜分?

吗啉环和哌嗪环类新衍生物对血管舒张功能的影响

目的 利用ETA与M受体的相似性和差异性 ,设计合成吗啉环和哌嗪环类新结构化合物 ,分析其对血管舒张功能的影响。方法 采用离体大鼠胸主动脉环 ,观察化合物对血管张力的影响 ;并观察L NAME ,吲哚美辛和阿托品对血管环最大舒张率的影响。结果 81个化合物中有 57个能舒张血管 ,其中 8个最大舒张率在 50 %～ 85 % ;活性化合物按母核可分为 8类 ;DMHPPP和PPVP诱发的内皮依赖性舒血管反应可被L NAME和吲哚美辛所拮抗 ,但不被阿托品拮抗 ;DMHPPP和PPVP还能显著增强乙酰胆碱 (ACh)诱发的内皮依赖性舒血管反应的最大舒张率。结论 具有舒血管作用的活性化合物通过促进内皮细胞释放一氧化氮(NO)和前列环素 (PGI2 )共同实现舒张效应 ;

新型莨菪烷类化合物对豚鼠回肠肌M受体动力学研究

目的探讨定向合成新型莨菪烷类化合物对豚鼠回肠肌Ｍ受体动力学的作用。方法采用豚鼠离体回肠纵肌累积剂量－效应曲线法，求得ｐＤ２（解离常数负对数）及Ｅｍａｘ（最大效应）值，以乙酰胆碱（ＡＣｈ为标准品，比较其亲和力和内在活性。结果ＭＡＣ－６、ＭＡＣ－７、ＭＡＣ－８、ＭＡＣ－１８和ＭＡＣ－１９化合物与豚鼠肠纵肌Ｍ受体具有不同程度的亲和力（ｐＤ２＝６．５９～８．７７），引起豚鼠肠纵肌收缩最大效应为ＡＣｈ的２９％～５７％，选择性Ｍ受体拮抗药阿托品能阻断其作用；相反，ＭＡＣ－１６能显著抑制由ＡＣｈ引起豚鼠肠纵肌收缩，呈竞争性拮抗作用，其ｐＡ２为７．７２。结论定向合成莨菪烷类化合物具有明显的Ｍ受体激动或阻断作用，并显示明显的构效关系特征。

鞘内注射M受体和GDNF反义寡脱氧核苷酸抑制吗啡戒断大鼠蓝斑c-Fos表达

应用免疫组化方法观察鞘内注射毒蕈碱型乙酰胆碱(muscarinic acetylcholine receptor,M) 受体和胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)反义寡脱氧核苷酸对吗啡戒断大鼠蓝斑(locus coeruleus,LC)区内Fos表达的影响。结果显示,鞘内注射M_2受体和GDNF反义寡脱氧核苷酸明显减少大鼠吗啡戒断症状评分值(n=6,P<0.05)。正常大鼠LC区神经元Fos基础表达较低,吗啡依赖大鼠LC区神经元Fos表达增加,吗啡依赖大鼠纳酪酮(4mg/Kg,ip)催促戒断后,Fos表达进一步增加;鞘内注射M_2受体和GDNF反义寡脱氧核苷酸处理后均减少吗啡戒断大鼠LC区神经元Fos表达(n=5,P<0.05)。而鞘内注射M_1受体反义寡脱氧核苷酸处理组LC 区神经元Fos表达较吗啡戒断组没有显著差异(n=5,P>O.05)。结果提示:脊髓M_2受体调节吗啡戒断时LC区的神经元激活,而这种神经上行性激活涉及神经元与胶质细胞之间的适应性调节。