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 Nov;38(7):701-12.
Stimulation of muscarinic receptors increases phosphoinositide (PI) hydrolysis in 132-1N1 human astrocytoma cells. To evaluate the subtype of receptors which mediate PI hydrolysis in 132-1N1 cells, the effects of: a) the nonselective M1 agonist, carbachol; b) the selective M1 agonist, 4-hydroxy-2-butynyl-trimethylammonium chloride-m-chlorocarbinilate (McN-343); c) the nonselective antagonists, atropine and scopolamine; d) the relatively selective M1 antagonist, pirenzepine; e) the relatively selective M2 antagonists, AF-DX 116 (11-2-diethylaminomethyl-1-piperidinylacetyl-5, 11-dihydro-6H-pyrido-2,3-b-1,4-benzodiazepine-6-one) and methoctramine and f) the relatively selective M3 antagonist, hexahydrosila-difenidol (HHSiD) on PI hydrolysis in 132-1N1 cells were studied. The cell pools of inositol-phospholipids were prelabelled by incubating 132-1N1 cells in a low inositol containing medium (CMRL-1066) supplemented with [3H]inositol (2 microCi/ml) for 20-24 hours at 37 degrees C. The cells were washed and resuspended in a physiological salt solution, and PI hydrolysis was measured by accumulation of [3H]inositol-1-phosphate (IP) in the presence of 10 mM LiCl. Carbachol produced time and concentration dependent PI hydrolysis (EC50, 37 microM). McN-A343 did not cause significant hydrolysis of PI in 132-1N1 cells indicating that the receptor was not of M1 type. All the above muscarinic antagonists caused a concentration dependent decrease in the level of IP in response to carbachol (100 microM). The rank order of their affinities (pA2 values) was: atropine (8.8) > HHSiD (7.6) > pirenzepine (6.8) > methoctramine (6.0) > AF-DX 116 (5.8). This rank order supports the concept that M3 (other names, M2 beta, glandular M2) receptors are linked to PI hydrolysis in 132-1N1 cells. HHSiD, which is selective for M3 receptors of the smooth muscle has higher affinity for muscarinic receptors in 132-1N1 cells than AF-DX 116 which is selective for M2 receptors in cardiac tissue. If the receptor in 132-1N1 cells had been M2, part of the rank order for affinities would have been methoctramine > AF-DX 116 > HHSiD > pirenzepine. From all of these observations, the muscarinic receptor for PI hydrolysis in 132-1N1 cells is tentatively characterized as of M3 type.
刺激毒蕈碱受体可增加132 - 1N1人星形细胞瘤细胞中的磷酸肌醇（PI）水解。为评估介导132 - 1N1细胞中PI水解的受体亚型，研究了以下物质的作用：a）非选择性M1激动剂卡巴胆碱；b）选择性M1激动剂4 - 羟基 - 2 - 丁炔基 - 三甲基氯化铵 - 间氯卡宾酯（McN - 343）；c）非选择性拮抗剂阿托品和东莨菪碱；d）相对选择性M1拮抗剂哌仑西平；e）相对选择性M2拮抗剂AF - DX 116（11 - 2 - 二乙氨基甲基 - 1 - 哌啶基乙酰基 - 5,11 - 二氢 - 6H - 吡啶并[2,3 - b][1,4]苯并二氮杂䓬 - 6 - 酮）和甲奥氮平；f）相对选择性M3拮抗剂六氢硅二苯地洛（HHSiD）对132 - 1N1细胞中PI水解的影响。通过在含[3H]肌醇（2微居里/毫升）的低肌醇培养基（CMRL - 1066）中于37℃孵育132 - 1N1细胞20 - 24小时，对肌醇磷脂的细胞池进行预标记。细胞经洗涤后重悬于生理盐溶液中，在10 mM LiCl存在下，通过[3H]肌醇 - 1 - 磷酸（IP）的积累来测量PI水解。卡巴胆碱引起时间和浓度依赖性的PI水解（EC50为37 microM）。McN - A343在132 - 1N1细胞中未引起显著的PI水解，表明该受体不是M1型。所有上述毒蕈碱拮抗剂均导致对卡巴胆碱（100 microM）反应的IP水平呈浓度依赖性降低。它们的亲和力（pA2值）的顺序为：阿托品（8.8）> HHSiD（7.6）>哌仑西平（6.8）>甲奥氮平（6.0）> AF - DX 116（5.8）。该顺序支持了M3（其他名称，M2β，腺体M2）受体与132 - 1N1细胞中的PI水解相关的概念。对平滑肌M3受体具有选择性的HHSiD对132 - 1N1细胞中毒蕈碱受体的亲和力高于对心脏组织中M2受体具有选择性的AF - DX 116。如果132 - 1N1细胞中的受体是M2型，亲和力顺序的一部分将是甲奥氮平> AF - DX 116> HHSiD>哌仑西平。基于所有这些观察结果，132 - 1N1细胞中用于PI水解的毒蕈碱受体初步被鉴定为M3型。
