Dai Y S, Ambudkar I S, Horn V J, Yeh C K, Kousvelari E E, Wall S J, Li M, Yasuda R P, Wolfe B B, Baum B J
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Am J Physiol. 1991 Dec;261(6 Pt 1):C1063-73. doi: 10.1152/ajpcell.1991.261.6.C1063.
The binding affinities of muscarinic antagonists were compared with their abilities to block carbachol (CCh)-mediated stimulation of Ca2+ mobilization and inhibition of isoproterenol-elicited adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in rat parotid cells. The binding of [3H]quinuclidinyl benzilate (QNB) to membranes was inhibited by antagonists with the following potencies (dissociation constant, nM): atropine (1.1) approximately 4-diphenylacetoxy-N-methylpiperidine methbromide (4-DAMP) (1.6) much greater than pirenzepine (136) greater than 11-[[2-[(diethylamino)methyl-1-piperidinyl]-acetyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116) (5,293). AF-DX 116 blocked Ca2+ mobilization and inhibition of cAMP accumulation with low affinities [inhibitory concentration at 50% (IC50) = 3150 and 6,528 nM, respectively], whereas 4-DAMP blocked these responses with considerably higher affinities (IC50 = 4.3 and 11.4 nM, respectively). Schild plots of 4-DAMP and AF-DX 116 antagonism of CCh-stimulated inositol trisphosphate accumulation showed inhibitor constant (Ki) values of 0.85 and 1,585 nM, respectively, whereas Schild plots of 4-DAMP, AF-DX 116, and methoctramine antagonism of CCh-induced inhibition of cAMP accumulation showed Ki values of 1.3, 1,585, and 2,754 nM, respectively. Preincubation of cells with 0.1 mM 3-isobutyl-1-methylxanthine did not prevent the capacity of CCh to inhibit cAMP accumulation. Pertussis toxin blocked the CCh-elicited and Gi-mediated inhibition of cAMP formation. Northern blot analysis showed the presence of mRNA for the M3, but not for the M2, subtype in parotid gland. An immunochemical procedure using m1-m5 specific antibodies was performed in parotid membranes and showed that the m3 receptor accounts for 93% of precipitable receptors. These data suggest that M3 receptors in the rat parotid are coupled to both the stimulation of Ca2+ mobilization and the inhibition of cAMP accumulation.
比较了毒蕈碱拮抗剂的结合亲和力与其阻断卡巴胆碱（CCh）介导的大鼠腮腺细胞钙离子动员刺激以及抑制异丙肾上腺素引发的3',5'-环磷酸腺苷（cAMP）积累的能力。[3H]喹核醇基苯甲酸酯（QNB）与膜的结合被拮抗剂抑制，其效力如下（解离常数，nM）：阿托品（1.1）约等于4-二苯基乙酰氧基-N-甲基哌啶甲基溴化物（4-DAMP）（1.6）远大于哌仑西平（136）大于11-[[2-[(二乙氨基)甲基-1-哌啶基]-乙酰基]乙酰基]-5,11-二氢-6H-吡啶并[2,3-b][1,4]-苯并二氮杂卓-6-酮（AF-DX 116）（5293）。AF-DX 116以低亲和力阻断钙离子动员和cAMP积累的抑制作用[50%抑制浓度（IC50）分别为3150和6528 nM]，而4-DAMP以相当高的亲和力阻断这些反应（IC50分别为4.3和11.4 nM）。4-DAMP和AF-DX 116对CCh刺激的肌醇三磷酸积累的拮抗作用的Schild图显示抑制剂常数（Ki）值分别为0.85和1585 nM，而4-DAMP、AF-DX 116和甲奥克明对CCh诱导的cAMP积累抑制作用的拮抗作用的Schild图显示Ki值分别为1.3、1585和2754 nM。用0.1 mM 3-异丁基-1-甲基黄嘌呤预孵育细胞并不能阻止CCh抑制cAMP积累的能力。百日咳毒素阻断了CCh引发的以及Gi介导的cAMP形成的抑制作用。Northern印迹分析显示腮腺中存在M3亚型的mRNA，但不存在M2亚型的mRNA。在腮腺膜中使用m1 - m5特异性抗体进行免疫化学检测，结果显示m3受体占可沉淀受体的93%。这些数据表明大鼠腮腺中的M3受体与钙离子动员的刺激以及cAMP积累的抑制均相关。
