1,2,5,6-4H-1-烷基-3-取代吡啶类新衍生物对血管内皮细胞功能的调节作用及其构效关系

目的：以槟榔碱为结构母核，设计合成1，2，5，6-4H-1-烷基-3-取代吡啶类新衍生物，分析其对血管内皮细胞功能的影响。方法：采用离体大鼠主动脉环和离体豚鼠回肠收缩实验，分别观察新结构化合物对血管和回肠平滑肌张力的影响；并进一步观察一氧化氮合酶抑制剂L-NAME、环氧酶抑制剂吲哚美辛、M受体亚型非选择性激动剂毛果芸香碱和M受体亚型非选择性拮抗剂阿托品对新结构化合物诱发的血管舒张反应的影响。结果：从100个新化合物中发现4个有舒血管反应，但不激活M受体的化合物，分别是HH91、HH95、HH98、HH103。新化合物HH103诱发的内皮依赖性舒血管反应可被L-NAME所拮抗，但不被吲哚美辛、阿托品和毛果芸香碱拮抗。结论：新化合物可诱发内皮依赖性舒张反应并具有特定构效关系；HH103通过促进内皮细胞释放NO发挥其效应；但其作用特征又与乙酰胆碱不同。     

血管内皮细胞乙酰胆碱激活蛋白

内皮细胞乙酰胆碱激活蛋白 (EPA)介导的内皮依赖性血管舒张反应 ,广泛存在于哺乳动物的大小动脉血管。在心血管常见病与多发病中 ,EPA功能发生明显变化。EPA虽然具有经典M受体的某些特征 ,又与M受体不同 ,EPA不被毛果芸香碱激活 ,nmol·L-1阿托品不能取代EPA上乙酰胆碱的结合位点。EPA的内源性配体、分子药理学特征和受体类型有待于进一步研究     

氨甲酰胆碱和毛果芸香碱对血管内皮细胞M受体基因表达的调节作用

目的：比较氨甲酰胆碱和毛果芸香碱对血管内皮细胞M受体基因表达调节作用的异同点。方法：氨甲酰胆碱和毛果芸香碱分别孵育培养的牛主动脉内皮细胞10h后，提取细胞总RNA，RT-PCR方法测定M受体5个亚型mRNA的表达。结果：M_1～M_5受体mRNA在牛主动脉内皮细胞上均有表达，氨甲酰胆碱和毛果芸香碱孵育后表达量均增加，但二者之间无差异。结论：氨甲酰胆碱和毛果芸香碱对M受体5个亚型的基因表达均发生了相同的影响，两者对M受体可能的作用尚不能解释其对血管张力的影响。     

激活血管内皮细胞乙酰胆碱作用靶标的抗血栓作用及其分子机制

目的　在角叉菜胶诱发的小鼠尾动脉血栓模型上 ,研究激活血管内皮细胞乙酰胆碱作用靶标对血栓形成的影响并探讨其可能的分子机制。方法　比较既可激活血管内皮乙酰胆碱作用靶标 ,又可激活M受体的槟榔碱和仅可激活M受体的毛果芸香碱对成栓率和血栓长度影响的差异 ,分析激活内皮乙酰胆碱作用靶标对血栓的影响 ;通过测定血小板最大聚集率 ;凝血酶原时间 (PT)、活化部分凝血活酶时间(KPTT)和凝血酶时间 (TT) ;血浆中组织型纤溶酶原激活物(t PA)的含量和纤溶酶原激活物抑制剂 (PAI 1)的活性 ;血浆血栓素A2 (TXA2 )和前列环素 (PGI2 )含量 ,分析其可能的分子机制。结果　槟榔碱在 0 1～ 4 0mg·kg-1(ip)可剂量依赖性地对抗血栓形成 ,而在相同条件下 ,大剂量 (4mg·kg-1)的毛果芸香碱仍无抗血栓作用 ;槟榔碱对TT ,PT和KPTT和血小板最大聚集率 (MAR)均无影响 ;使血浆中t PA的含量升高 ,PAI 1的活性降低 ;使血栓形成过程中升高的TXA2 降低 ,并呈明显的量效关系 ,使血栓形成过程中降低的PGI2 升高。结论　激活血管内皮细胞乙酰胆碱靶标可对抗血栓形成 ,其可能的分子机制不通过直接影响凝血系统和血小板的聚集 ,而通过促进内皮细胞释放t PA和抑制内皮细胞合成并释放的PAI 1的活性 ,间接激活纤溶系统 ,发挥抗血栓?     

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 ;并可调节血管内皮细胞乙酰胆碱靶标的功能     

毛果芸香碱对哇巴因诱发豚鼠心律失常的保护作用

目的 观察毛果芸香碱对实验性心律失常模型的影响.方法 以哇巴因制备实验性动物心律失常模型,观察毛果芸香碱(0.2 mg· kg-1)的干预作用.结果 毛果芸香碱能明显延长哇巴因引起豚鼠出现心律失常后的存活时间(P＜0.05).毛果芸香碱的作用可被M3受体阻断剂4-DAMP(4-二苯乙酰氧-N-甲基哌啶甲碘化物)完全逆转.结论 毛果芸香碱通过激动豚鼠心肌M3受体而具有对抗哇巴因诱发豚鼠心律失常的作用,提示毛果芸香碱有良好的抗心律失常作用.     

激活乙酰胆碱作用靶标对血管内皮细胞基因表达的影响

目的　研究激活乙酰胆碱作用靶标 (ETA)对血管内皮细胞基因表达的影响。方法　采用人类cDNA表达谱芯片 ,比较氨甲酰胆碱和毛果芸香碱对人冠脉内皮细胞基因表达的影响及其生物学功能。结果　既激活ETA又激活M受体的氨甲酰胆碱 ,以及仅激活M受体的毛果芸香碱 ,在 10 0μmol·L-1浓度下与内皮细胞共孵育 10h ,以含有 14 0 0 0条人类unigene的BiostarH 14 112ScDNA表达谱芯片检测内皮细胞基因表达的变化 ,发现差异基因共 80 1条。两药共同诱导的差异基因 4 91条 ,上调 2 0 5条 ,下调 2 86条。两药诱导差异基因存在很大差别 ,其中氨甲酰胆碱诱导上调基因 119条 ,下调基因 191条 ,这些差异基因均不受毛果芸香碱的影响。进一步分析氨甲酰胆碱特异性诱导差异基因的功能 ,发现有受体与G蛋白、离子通道、血栓、动脉粥样硬化相关基因等七大类。结论　氨甲酰胆碱特异性诱导的差异基因与ETA及其效应器系统以及激活ETA产生抗动脉粥样硬化和抗血栓的分子机制相关。     

毛果芸香碱对心律失常保护作用的研究

目的观察毛果芸香碱对动物心律失常模型的作用。方法分别以乌头碱和氯化钡制造动物心律失常模型,观察毛果芸香碱(0.2mg/kg)对心律失常的影响。结果毛果芸香碱可明显延迟乌头碱引起的大鼠室性心律失常的出现(P<0.05);延长大鼠出现心律失常后的存活时间(P<0.05);毛果芸香碱可明显延迟氯化钡引起的大鼠双向室性心律失常的出现(P<0.05)、缩短大鼠心律失常的持续时间(P<0.05)。4-DAMP可完全拮抗毛果芸香碱诱导的心律失常。结论毛果芸香碱通过激动大鼠心肌M3受体而产生对抗乌头碱和氯化钡诱发大鼠心律失常的作用。     

毛果芸香碱对实验性心律失常的保护作用

目的：观察毛果芸香碱对实验性心率失常的影响。方法：分别以乌头碱，氯化钡和哇巴因制备实验性动物心律失常模型，观察毛果芸香碱的干预作用。结果：毛果芸香碱可显著延迟乌头碱引起的大鼠室性心律失常的出现（P〈0．05），延长出现心律失常后的存活时间（P〈0．05）；能明显延迟氯化钡引起的大鼠双相室性心律失常的出现（P〈0．01），缩短心律失常的持续时间（P〈0．01）；能显著延长哇巴因引起豚鼠出现心律失常后的存活时间（P〈0．05）。毛果芸香碱的上述作用可被M3受体阻断荆4-DAMP完全逆转。结论：毛果芸香碱具有对抗乌头碱和氯化钡诱发大鼠，哇巴因诱发豚鼠心律失常的作用，提示其具有良好的抗心律失常作用；毛果芸香碱通过激动大鼠和豚鼠心肌M3受体而产生抗心律失常的作用。     

血管内皮细胞非神经性M受体药理学与疾病治疗的研究进展

血管内皮细胞上存在介导内皮依赖性血管舒张反应的血管内皮细胞非神经性毒蕈碱样受体(M受体)。对血管内皮细胞非神经性M受体亚型的定性一直存在争议。多数药理实验结果显示血管舒张反应是M3受体介导的;另外M1,M2受体也在不同组织类型的血管上介导了血管舒张反应。基因敲除小鼠的结果显示,主动脉、冠状动脉上介导舒张反应的受体以M3亚型为主;脑动脉上以M5为主。药理实验发现,介导血管舒张效应的内皮细胞非神经性M受体具有相对的组织、器官选择性和动物种属差异,与神经性M受体药理学特性有差别。激活血管内皮细胞非神经性M受体可促使内皮细胞分泌多种血管活性因子,介导血管的舒张、维持血管的正常生理状态;同时还可促进组织型纤溶酶原激活物的生成,降低多种粘附分子的分泌,产生抗动脉粥样硬化和抗血栓形成的功能。与传统药物作用机制不同。     

Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only.     

Muscarinic receptor subtypes mediating vasorelaxation of the perforating branch of the human internal mammary artery

The effect of acetylcholine (ACh) on the isolated perforating branch of the human internal mammary artery (HIMA) was investigated. ACh induced concentration- and endothelium-dependent relaxation of arterial rings precontracted with phenylephrine (pEC(50) = 6.93 +/- 0.01). The muscarinic receptor antagonist atropine (no selectivity), pirenzepine (M(1)), methoctramine (M(2)), and p-fluoro-hexahydro-siladifenidol (M(1)/M(3)) competitively antagonized the response to ACh. The pA(2) values were 9.81 +/- 0.15, 7.74 +/- 0.08, 6.27 +/- 0.08, and 7.88 +/- 0.04, respectively. In conclusion, this study has shown that ACh induced an endothelium-dependent relaxation of the perforating branch of the HIMA by stimulation of muscarinic receptors on the endothelial cells. On the basis of differential antagonist affinity, we suggest that the muscarinic receptors involved in the ACh-induced relaxation of the isolated perforating branch of HIMA are predominantly of M(1) subtype.     

The affinity of m-cholinomimetics for the m-cholinoreceptors in different tissues

The affinities of some cholinomimetics (acetylcholine, methacholine, carbachol, oxotremorine, aceclidine and pilocarpine) for muscarinic acetylcholine receptors of various isolated tissues (rat spontaneously beating atria, rat electrically stimulated left atrium, segment of rat aorta precontracted by PGF2 alpha, rat trachea and guinea pig trachea) was estimated in functional experiments. It was established that each of full agonists (acetylcholine, methacholine, carbachol, oxotremorine and aceclidine) has lower affinities for muscarinic M2 receptors of rat atria as compared with its affinity for muscarinic M3 receptors of rat aorta. The partial agonist pilocarpine has similar affinities for muscarinic receptors of rat atria and rat aorta. It was shown that contractile response of guinea pig tracheal smooth muscle is two-staged. It was found that rat and guinea pig tracheal smooth muscle contractions are mediated primarily by muscarinic M3 cholinoceptors and partially by M2 cholinoceptors.     

Evidence for prejunctional M2 muscarinic receptors in pulmonary cholinergic nerves in the rat.

1. The effects of muscarinic antagonists considered to be selective for M1 receptors (pirenzepine) and for M2 receptors (gallamine and methoctramine) were used to investigate the existence of prejunctional muscarinic receptors on cholinergic nerves in the rat lung. The tracheal tube preparation was used in vitro, and contraction of the trachealis muscle was induced by electrical field stimulation (EFS) and by application of an exogenous muscarinic agonist (pilocarpine), and measured as an increase in intraluminal pressure in the tube. 2. The muscarinic antagonists, gallamine and methoctramine, enhanced the contractions induced by nerve stimulation, while contractions elicited by exogenous application of pilocarpine were inhibited by the antagonists. 3. In contrast, pirenzepine blocked contractions induced by both EFS and pilocarpine in a dose-dependent manner (EC50 0.1 microM) due to blockade of the postjunctional muscarinic receptors on airway smooth muscle. Potentiation of the response to EFS was never seen with this antagonist. 4. The muscarinic agonist, pilocarpine, caused a slow maintained increase in tone of the tracheal tube and at the same time reduced the contractions induced by EFS. This inhibitory effect was blocked by gallamine and methoctramine. 5. The results suggest that prejunctional inhibitory muscarinic receptors may be localised on the parasympathetic cholinergic nerve terminals innervating tracheal smooth muscle in the rat. This confirms previous findings obtained by measuring transmitter release in this species. The present results suggest that these receptors are of the M2 subtype. Blockade of these autoreceptors with gallamine or methoctramine would increase the output of acetylcholine (ACh) and thereby enhance the nerve-induced contraction of tracheal smooth muscle.     

p-Fluoro-hexahydro-sila-difenidol: affinity for vascular muscarinic receptors

The M3-selective antagonist, p-fluorohexahydro-sila-difenidol was used to characterize muscarinic receptors in two vascular preparations, the rabbit ear artery with an endothelium-dependent relaxation and the bovine coronary artery with an endothelium-independent contractile response. pKB values were consistent with the presence of M3 receptors, 7.9 and 7.5 in coronary and ear arteries, respectively. These findings confirm that muscarinic receptors of the rabbit ear artery and bovine coronary artery have similar characteristics and belong to the M3 subtype.     

山莨菪碱对乙酰胆碱诱导的猫离体动脉内皮依赖性舒张反应的影响(英文)

目的　观察山莨菪碱对乙酰胆碱 (ACh)诱导的内皮依赖性血管舒张反应的影响。方法　采用猫离体血管功能实验 ,观察山莨菪碱对ACh诱发的内皮依赖性血管舒张反应的影响。结果　在猫肠系膜动脉、肾动脉和股动脉 ,山莨菪碱 0 .0 1～ 1 .0nmol·L-1 能够浓度依赖地抑制ACh诱导的内皮依赖的血管舒张反应。山莨菪碱抑制 1 0 μmol·L-1 ACh所诱导血管舒张的IC50 分别为 0 .2 36 ,0 .72 9和 0 .50 8nmol·L-1 ,山莨菪碱的拮抗作用符合非竞争性拮抗模式。此外 ,1 0nmol·L-1 山莨菪碱能有效拮抗ACh诱导的冠状动脉内皮依赖性舒张反应。结论　山莨菪碱能强效拮抗ACh诱发的内皮依赖性血管舒张反应 ,这种效应具有组织特异性的特点。     

Increased function of inhibitory neuronal M2 muscarinic receptors in trachea and ileum of diabetic rats

1. Release of acetylcholine from parasympathetic nerves is inhibited by neuronal M(2) muscarinic receptors. The effects of streptozotocin-induced diabetes on prejunctional M(2) and postjunctional M(3) muscarinic receptor function in rat trachea and ileum were investigated in vitro. 2. Neuronal M(2) muscarinic receptor function was tested by measuring the ability of an agonist, pilocarpine, to inhibit and an antagonist, methoctramine, to potentiate electrical field stimulation (EFS)-induced contraction of trachea and ileum. Concentration-response curves to pilocarpine and methoctramine were shifted to the left in both to a greater degree in diabetics than controls. 3. In trachea, post-junctional M(3) muscarinic receptor function was increased since maximum contractile responses to the muscarinic agonists acetylcholine and carbachol were greater in diabetics than controls. This increase offset the increased function of the inhibitory neuronal M(2) muscarinic receptors since EFS-induced, frequency-dependent contraction was equal in control and diabetic rats. 4. In contrast, post-junctional M(3) muscarinic receptor function was unchanged by diabetes since concentration-response curves to acetylcholine and carbachol were not different between groups. Thus, EFS-induced contractions of the ileum were decreased in diabetics versus controls. 5. In conclusion, inhibitory M(2) muscarinic receptors on parasympathetic nerves in the trachea and ileum are hyperfunctional in diabetic rats. The function of post-junctional M(3) muscarinic receptors in the trachea, but not the ileum, is also increased in diabetes. 6. The dysfunction of inhibitory, neuronal M(2) muscarinic receptors in the airways may protect against hyperreactivity and in the ileum may contribute to gastrointestinal dysmotility associated with diabetes.     

An autoradiographic study of muscarinic cholinoceptors in blood vessels: no localization on vascular endothelium

In vitro labelling and autoradiographic techniques were used to examine the localization of [3H]quinuclidinyl benzilate ([3H]QNB) and [125I]4-iodo-QNB ([125I]4IQNB) to slide-mounted sections of rabbit aorta and pulmonary artery, cat aorta, pulmonary and superior mesenteric arteries. These vessels all respond to acetylcholine (ACh) with endothelium-dependent relaxation, yet there was no evidence for endothelium-related binding of either [3H]QNB or [125I]4IQNB. Muscarinic receptors were localized over the medial smooth muscle and, in the rabbit pulmonary artery, the density of binding increased towards the adventitia. Binding of either radioligand to sections of rabbit pulmonary artery was not affected by the muscarinic M1 receptor antagonist pirenzepine (20 nM) but was markedly reduced by the muscarinic M2 antagonist 4DAMP (4-diphenylacetoxy-N-methyl-piperidine methobromide) (1 nM). This study provides evidence for muscarinic receptors located directly on smooth muscle cells, indicating that endothelium-dependent relaxation to ACh results from an indirect mechanism involving smooth muscle muscarinic receptors.     

Inhibition of neuronal M(2) muscarinic receptor function in the lungs by extracellular nitric oxide

1. These experiments were carried out to test whether neuronal M(2) muscarinic receptor function in the lungs is affected by nitric oxide (NO) and whether the source of the NO is epithelial or neuronal. 2. In pathogen free, anaesthetized guinea-pigs, the muscarinic agonist pilocarpine inhibited vagally induced bronchoconstriction demonstrating functional neuronal M(2) muscarinic receptors. In the presence of the NO donor, 3-morpholino-sydnonimine (SIN-1), pilocarpine no longer inhibited vagally induced bronchoconstriction. In contrast, inhibiting endogenous NO with N(G)-monomethyl-L-arginine methyl ester (L-NMMA) did not affect the ability of pilocarpine to decrease vagally induced bronchoconstriction. 3. In isolated tracheas, pilocarpine inhibited contractions induced by electrical field stimulation demonstrating that neuronal M(2) muscarinic receptors function in vitro. As in the anaesthetized guinea-pigs, SIN-1 shifted the pilocarpine dose response curve to the right, demonstrating decreased neuronal M(2) receptor function. However, in vitro, L-NMMA shifted the pilocarpine dose response curve to the left, demonstrating that endogenous NO was inhibiting the ability of the M(2) receptors to decrease acetylcholine (ACh) release. 4. Both haemoglobin (Hb), which scavenges NO, and epithelial removal also shifted the pilocarpine dose response curve to the left, demonstrating that the NO inhibiting neuronal M(2) receptor function was extracellular and probably of epithelial origin. 5. In conclusion, extracellular NO appears to inhibit the ability of the M(2) receptors to decrease ACh release from the parasympathetic nerves in the lungs in vivo and in vitro in pathogen free guinea-pigs. However, while the neuronal M(2) receptors will respond to NO (from SIN-1) in vivo, there does not appear to be an endogenous source of NO since L-NMMA had no effect in vivo.     

Mediation by M3-muscarinic receptors of both endothelium-dependent contraction and relaxation to acetylcholine in the aorta of the spontaneously hypertensive rat.

1. Experiments were designed to characterize the subtype(s) of endothelial muscarinic receptor that mediate(s) endothelium-dependent relaxation and contraction in the aorta of spontaneously hypertensive rats (SHR). 2. Rings of SHR aorta with endothelium were suspended in organ baths for the measurement of isometric force. Ecothiopate (an inhibitor of acetylcholinesterase) was present throughout the experiments. Endothelium-dependent contraction to acetylcholine was studied in quiescent aortic rings in the presence of NG-nitro-L-arginine (to prevent the formation of nitric oxide). Endothelium-dependent relaxation to acetylcholine was obtained during contraction to phenylephrine and in the presence of indomethacin (to inhibit cyclo-oxygenase activity). Responses to acetylcholine were assessed against the non-preferential muscarinic receptor antagonist, atropine, and the preferential antagonists pirenzepine (M1), methoctramine (M2) and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP; M3). 3. The potency of acetylcholine in inducing endothelium-dependent contraction was 6.54 +/- 0.07 (EC50). Atropine, pirenzepine, methoctramine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent contraction to acetylcholine. The pA2 values for these muscarinic receptor antagonists were estimated from Arunlakshana-Schild plots to be (-log M) 9.48 +/- 0.07, 6.74 +/- 0.22, 6.30 +/- 0.20 and 9.39 +/- 0.22 respectively. The potency of acetylcholine in inducing endothelium-dependent relaxation was 7.82 +/- 0.09 (IC50). Atropine, pirenzepine and 4-DAMP displayed competitive antagonism towards the endothelium-dependent relaxation to acetylcholine but methoctramine had no effect. The pA2 values for atropine and 4-DAMP for the relaxation to acetylcholine were estimated from Arunlakshana-Schild plots to be (-log M) 9.15 +/- 0.23 and 9.63 +/- 0.28, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)