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。     

激活血管内皮细胞非神经性M受体对不同亚型G蛋白基因表达的影响

目的研究G蛋白各亚型在内皮细胞上的表达分布,以及氨甲酰胆碱和毛果芸香碱的调节作用。方法培养大鼠主动脉内皮细胞,用氨甲酰胆碱和毛果芸香碱10-4mol.L-1分别孵育,6 h后提取细胞总RNA,RT-PCR方法测定G蛋白不同亚型mRNA的表达。结果G蛋白4个亚家族中的Gq/11、Gs、G i在内皮细胞有表达,而G12/13未见表达,氨甲酰胆碱和毛果芸香碱分别作用6 h后,Gs、G i家族、Gq/11家族中的G11蛋白基因表达均未发生变化,只有Gq/11家族中的Gq蛋白在氨甲酰胆碱10-4mol.L-1作用下,基因表达上调了72.7%,而相同浓度的毛果芸香碱却不影响Gq蛋白的基因表达。结论氨甲酰胆碱激活NNMR后,G蛋白4个亚家族中只有Gq/11家族中的Gq蛋白基因表达发生了变化,可以推测Gq蛋白在NNMR的信号转导中发挥了主要的作用。     

M_3受体激动剂对氯化钡诱发大鼠心律失常的保护作用

目的观察M3受体激动剂胆碱和毛果芸香碱对氯化钡诱发Wistar大鼠心律失常的保护作用。方法舌下静脉注射氯化钡诱发Wistar大鼠心律失常,分别给予胆碱和毛果芸香碱以及M3受体特异阻断剂4-二苯乙酰氧-N-甲基哌啶甲碘化物(4-DAMP)干预,观察大鼠心律失常的发生和发展情况。结果氯化钡4mg·kg-1可诱发大鼠出现明显的双向性室性心律失常,胆碱10mg·kg-1和毛果芸香碱0.2mg·kg-1均能推迟双向性室性心律失常的出现,并使心律失常的持续时间缩短(P<0.01)、严重程度减轻(P<0.01)。胆碱和毛果芸香碱的上述作用又被M3受体阻断剂4-DAMP0.12μg·kg-1部分阻断(P<0.05)。结论胆碱和毛果芸香碱通过激动心肌M3受体,对氯化钡诱发Wistar大鼠心律失常的发生和发展具有保护作用。     

内皮细胞乙酰胆碱靶标不同于M受体的药理学特征

目的 研究内皮细胞乙酰胆碱靶标与M受体药理学特征的差异。方法 采用离体血管环实验方法,观察抗M_3受体的单克隆抗体和M受体激动剂毛果芸香碱对乙酰胆碱引起的内皮依赖性舒张血管作用的影响。结果 毛果芸香碱能够剂量依赖性拮抗乙酰胆碱诱导的内皮依赖性血管舒张作用,其拮抗性质为非竞争性拮抗;抗M_3受体的单克隆抗体不影响乙酰胆碱诱发的内皮依赖性舒张血管作用,但能拮抗乙酰胆碱诱发的离休肠肌收缩作用。结论 内皮细胞乙酰胆碱靶标的药理学特征不同于经典M受体。     

利用基因芯片技术阐述甘草酸协同麻黄碱的分子作用机制

目的：利用基因表达谱芯片探索甘草酸协同麻黄碱对人支气管平滑肌细胞的协同作用机制。方法：采用TNF-Ⅱ诱导人支气管平滑肌细胞建立炎症模型,再分别用麻黄碱、麻黄碱联合甘草酸进行干预。提取细胞的mRNA,反转录后与基因表达谱芯片进行杂交,经扫描分析筛选表达差异的基因。结果：共筛选出显著表达差异基因53种,其中33种基因表达水平上调,20种基因表达下调,经GeneGo软件分析这些基因在不同生物途径中存在协同作用。结论：甘草酸在G蛋白偶联受体途径、细胞周期与凋亡以及离子通道等不同途径协同麻黄碱发挥作用。     

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.     

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。     

毛果芸香碱临床应用的新进展

毛果芸香碱(pilocarpine匹鲁卡品)是从毛果芸香类植物提取的一种生物碱。它属于拟胆碱药,主要作用于毒蕈碱样M受体。 药理作用 全身性作用 毛果芸香碱刺激外分泌腺的分泌,从而引起出汗、流泪、唾液分泌,以及胃液和胰液分泌增加。此外,还可兴奋肠道、泌尿道、膀胱、胆道和支气管、子宫等平滑肌,使其张力与能动性增加。本品po易吸收,主要通过肾脏排泄,T_(1/2)约1h。     

国内外抗青光眼药物简介

<正> 长期以来,抗青光眼药物以传统的毛果芸香碱为主。目前国内外已研制成功一些新的抗青光眼药物,按其作用机制不同,分以下3种主要类型试述。 1 使房水排出增多,而不影响房水生成酌药物 1.1拟胆碱药 (1)N-脱甲基氨甲酰胆碱(N-demethylated carbachel,DMC)本品     

Tissue-dependent association of muscarinic acetylcholine receptors with guanine nucleotide-binding regulatory proteins.

The muscarinic acetylcholine receptors in heart and cerebellum form a stable association with guanine nucleotide-binding regulatory proteins (G proteins) in the presence of receptor agonists. This has been confirmed by purification of the muscarinic receptor-G protein complexes using an immunoprecipitation protocol. The isolated complexes were subjected to Western blotting to identify the G protein subunits present in the complexes. At saturating concentrations of carbachol, the muscarinic receptors in atrial membranes co-purified exclusively with Go, whereas in cerebellar and ventricular membranes an association with both Gi and Go was demonstrated. Further characterization of the G protein subunits allowed identification of the species of Gi alpha subunits present in the complexes of muscarinic receptor and G protein; in ventricle Gi alpha 2 was the only subtype present, whereas in cerebellum both Gi alpha 1 and Gi alpha 2 were present. These results demonstrate that a single muscarinic receptor subtype, depending on the tissue studied, is capable of interacting with more than one G protein subtype. The concentrations of agonist required to promote receptor-G protein association in atrial and ventricular membranes correlated with the high affinity component of receptor occupancy by agonist, as measured in equilibrium binding assays. Furthermore, incubation of cardiac membranes with saturating concentrations of pilocarpine or McN A343 resulted in reduced amounts of receptor-G protein complexes, compared with carbachol. Overall, our results suggest that the specificity of cellular effects of muscarinic agonists may relate, in part, to the selective interaction of receptor with G proteins.     

Aging and cholinergic responses in bovine trachealis muscle.

1. The relative potencies of muscarinic agonists on bovine tracheal smooth muscle were unchanged as a consequence of aging and were carbachol greater than oxotremorine greater than muscarine greater than pilocarpine greater than McNeil A-343. 2. During aging, the potencies of carbachol, oxotremorine, McNeil A-343 and pilocarpine, but not muscarine, were reduced. 3. Maximal induced tensions to all the agents studied were reduced as a consequence of age. 4. Irreversible antagonism with benzilylcholine mustard showed that agonist efficacy was significantly reduced during aging. 5. Estimated receptor occupancy at the EC50 was significantly greater in tracheal tissues from the mature versus immature cows for every agonist studied. 6. The dissociation constants for full agonists (carbachol, oxotremorine and methacholine) were decreased with maturation while the converse was observed with partial agonists (McNeil A-343, pilocarpine). 7. We conclude that there are significant changes in the properties and coupling of muscarinic receptors during aging. These changes may contribute to the reduced airway reactivity seen in vivo.     

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.     

The SH-H subgroup of cardiac M2 receptors (M2 alpha) inhibits adenylate cyclase activity

Muscarinic receptors in the guinea-pig heart seem to consist entirely of M2 receptors, but are coupled with several responses including inhibition of adenylate cyclase activity. On the other hand three affinity states (SH, H and L) can be distinguished in cardiac membranes with muscarinic agonists such as carbachol. We showed previously that the three agonist binding states were the sum of two equilibria (SH-H and H-L subgroup), both regulated by GTP-binding protein(s). In this study we determined which subgroup was responsible for the inhibitory effect of muscarinic M2 receptors on adenylate cyclase activity. The ED50 values for this response of four muscarinic agonists, acetylcholine, carbachol, pilocarpine and oxotremorine corresponded with the binding KD values of H (acetylcholine and carbachol) and LO/P (pilocarpine and oxotremorine) sites. After alkylation of spare receptors, the ED50 value of carbachol was changed from 4.3 to 5.6 microM, which corresponded with the KD value of the H site. Furthermore, the four agonists were almost fully active when membrane preparations were pretreated with propylbenzilylcholine mustard (PrBCM) in the presence of carbachol to destroy the H-L subgroup, whereas after pretreatment with PrBCM and atropine, which alkylated both types of subgroups evenly, the decrease in the number of receptors was proportional to the decrease in the inhibitory effect on adenylate cyclase activity. These results suggest that only the SH-H subgroup (M2 alpha) is responsible for the inhibitory action of muscarinic receptors on adenylate cyclase activity in the heart.     

Apparent down-regulation of muscarinic acetylcholine receptors of neuroblastoma cells by pilocarpine is due to occluded agonist

Down-regulation of muscarinic acetylcholine receptors on N1E 115 neuroblastoma cells caused by muscarinic agonists has been studied. Whereas the potent agonist carbachol provokes a true down-regulation of receptors, the down-regulation caused by the partial agonist pilocarpine is only apparent and is actually due to the presence of pilocarpine taken up by the cells and released into the binding assays upon cell breakage.     

Muscarinic acetylcholine receptors linked to the inhibition of adenylate cyclase activity in membranes from the rat striatum and myocardium can be distinguished on the basis of agonist efficacy

Muscarinic agonists produce an inhibition of adenylate cyclase activity in broken cell preparations from rat striatum and myocardium. We have attempted to determine the occupancy-response relationships of three muscarinic agonists (carbachol, arecoline, and pilocarpine) by comparing their dose-response curves in these preparations with occupancy curves obtained under the same conditions. These comparisons suggest that the occupancy-response relationships for all three agonists in myocardium and for arecoline and pilocarpine in striatum are linear, response being directly proportional to occupancy. However, there appears to be a considerable receptor reserve for carbachol in the striatum, with carbachol producing a 50% maximal response at concentrations that occupy only 3% of the striatal receptors. These postulated occupancy-response relationships have been tested by blocking different proportions of the muscarinic receptors with the irreversible antagonist benzilylcholine mustard. The effects of this blockade on the dose-response curves are as predicted from the occupancy-response relationships, suggesting that these relationships are correct. The relative efficacies of these muscarinic agonists can be determined from their occupancy-response relationships. The efficacies of arecoline and pilocarpine relative to carbachol are approximately 0.13 and approximately 0.03 in striatum and approximately 1.0 and approximately 0.45 in myocardium, respectively. This difference in relative efficacies in the two tissues is evidence of a real conformational difference between the muscarinic receptors linked to adenylate cyclase in these preparations.     

Effects of cooling on the contractile response of the rat tracheal muscle to pilocarpine in vitro

Experiments were designed to determine the effect of cooling on the contractile response of rat tracheal strip-chain preparation to the partial agonist pilocarpine. The preparation was suspended in an organ bath containing Krebs bicarbonate solution for isometric tension recording. A decrease of the bath temperature from 37 to 30, 20 or 10 degrees C (cooling) did not affect or inhibited the contractile response of the trachea caused by pilocarpine (1-100 microM), while it augmented the carbachol (0.1-1 microM)-induced response. At 37 degrees C, the pilocarpine (3-100 microM)-induced contraction was relatively resistant to removal of extracellular Ca2+ or to the Ca2+ entry blocker, verapamil (1 microM). Similar results were also obtained for the carbachol (0.3-10 microM)-induced response. On the other hand, the affinity of pilocarpine for the tracheal muscarinic receptors, determined from its dissociation constant (KA), was significantly decreased at a lower temperature. It is concluded from these observations that cooling-induced subsensitivity of the rat tracheal muscle to pilocarpine is mainly associated with a decrease in the affinity of the agonist for muscarinic receptors.     

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.     

Muscarinic subsensitivity without receptor change in monkey ciliary muscle.

Intense muscarinic stimulation of the monkey ciliary muscle causes long-lasting muscarinic subsensitivity. This could be due to changes in number or affinity of muscarinic receptors which would cause a threshold elevation detectable in vivo. Since plasma levels of the agonists causing contraction in vivo were not available, the accommodation response to systemic muscarinic agents in subsensitized eyes was compared with that in the normal fellow eyes, usually seven days after a single subsensitizing dose of 100 micrograms carbachol to cornea. Subsensitivity was present whether the agonist tested was pilocarpine, carbachol or bethanechol but no evidence for threshold elevation was found. The conclusion is that changes in the muscarinic receptors are of minor importance for the kind of subsensitivity studied.