Stephan C C, Sastry B V
Department of Pharmacology, Vanderbilt University, School of Medicine, Nashville, TN 37232-2125.
Cell Mol Biol (Noisy-le-grand). 1992 Aug-Sep;38(5-6):601-12.
The subtype of muscarinic receptor which mediates cAMP attenuation is not established. Therefore, several selective muscarinic antagonists were used to characterize the subtype of muscarinic receptor coupled to the inhibition of hormone-stimulated cAMP accumulation using NG108-15 neuroblastoma x glioma hybrid cells. These cells were prelabeled with [2-3H]-adenine, washed, and resuspended in a culture medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM). The labeled cells were preincubated with the different antagonists 12-15 min. before they were challenged with agonists. The formation of [3H]-cAMP was activated by PGE1 (1 microM) or forskolin (1 microM). In all cases, [3H]-cAMP formed was separated and measured. Carbachol (100 microM) and McN-A343 (10 mM) were used as standard muscarinic agonists. These studies gave the following results: a) McN-A343 (10 mM), an M1 receptor agonist, was only a partial agonist causing 40% inhibition of cAMP accumulation indicating that this effect was not mediated by an M1 receptor; b) The M1-selective antagonist, pirenzepine, exhibited low affinity (pA2 6.2) further suggesting that an M1 receptor was not coupled to the attenuation of cAMP accumulation; c) Two selective M2 antagonists (AF-DX 116 and methoctramine) and M3 antagonist (HHSiD) were used to further characterize these muscarinic receptors. The order of all antagonists based on their affinities (pA2 values) could be arranged in the following order: atropine (9.0) > methoctramine (7.6) > HHSiD (6.9) > AF-DX 116 (6.6) > pirenzepine (6.2). HHSiD exhibits the same degree of affinity to M2 receptors of other tissues as it does to those of NG cells.(ABSTRACT TRUNCATED AT 250 WORDS)
介导环磷酸腺苷（cAMP）衰减的毒蕈碱受体亚型尚未明确。因此，使用几种选择性毒蕈碱拮抗剂，利用NG108 - 15神经母细胞瘤x胶质瘤杂交细胞来确定与激素刺激的cAMP积累抑制相关的毒蕈碱受体亚型。这些细胞先用[2 - 3H] - 腺嘌呤预标记，洗涤后，重悬于含有磷酸二酯酶抑制剂3 - 异丁基 - 1 - 甲基黄嘌呤（0.5 mM）的培养基中。标记后的细胞在受到激动剂刺激前，先与不同的拮抗剂预孵育12 - 15分钟。[3H] - cAMP的形成由前列腺素E1（1 microM）或福斯可林（1 microM）激活。在所有情况下，形成的[3H] - cAMP都被分离并测定。卡巴胆碱（100 microM）和McN - A343（10 mM）用作标准毒蕈碱激动剂。这些研究得出了以下结果：a）McN - A343（10 mM），一种M1受体激动剂，只是一种部分激动剂，导致cAMP积累抑制40%，表明这种效应不是由M1受体介导的；b）M1选择性拮抗剂哌仑西平表现出低亲和力（pA2 6.2），进一步表明M1受体与cAMP积累的衰减无关；c）两种选择性M2拮抗剂（AF - DX 116和甲溴东莨菪碱）和M3拮抗剂（HHSiD）被用于进一步确定这些毒蕈碱受体。所有拮抗剂基于其亲和力（pA2值）的顺序可排列如下：阿托品（9.0）>甲溴东莨菪碱（7.6）>HHSiD（6.9）>AF - DX 116（6.6）>哌仑西平（6.2）。HHSiD对其他组织的M2受体表现出与对NG细胞的M2受体相同程度的亲和力。（摘要截短于250字）
