Misle A J, Lippo de Bécemberg I, González de Alfonzo R, Alfonzo M J
Sección de Biomembranas, Instituto de Medicina Experimental (IME), Universidad Central de Venezuela (UCV), Caracas.
Biochem Pharmacol. 1994 Jul 5;48(1):191-5. doi: 10.1016/0006-2952(94)90239-9.
The binding of L-[benzilic-4,4'-3H]quinuclidinyl benzilate was studied in the plasma membrane fraction of bovine tracheal smooth muscle treated with the alkylating agent N-ethylmaleimide (NEM). It was found that NEM (2.5 mM) reduced significantly the Bmax from 1116 to 853 fmol/mg protein and increased the KD values of the muscarinic acetylcholine receptor (mAchR) activity from 36 to 61 pM. The mAchR subtypes in these plasma membranes were studied using competition experiments with selective antagonists. Pirenzepine displayed low competitive activity, having a pKi of 6.91 +/- 0.03, which was similar to that of AF-DX 116 (11[[2-[(diethylamino)methyl]- 1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3- b][1,4]benzodiazepine-6-one); (pKi = 6.90 +/- 0.04), whereas hexahydrodifenidol (HDD) and its p-fluoro-derivative (p-FHHSiD) showed higher affinities than pirenzepine, having pKi values of 7.45 +/- 0.05 and 7.17 +/- 0.06, respectively. The antagonist 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) showed a pKi of 8.25 +/- 0.03, which did not differ significantly from the affinity shown by methoctramine (pKi = 8.00 +/- 0.04). These data indicate that the mAchR associated with the plasma membrane fraction isolated from bovine airway smooth muscle can be classified as an M2 subtype muscarinic receptor. NEM treatment altered the affinities of the mAchR towards specific antagonists, such as methoctramine (Ki increased 3 times), and the results indicated that the alkylated mAchR behaves as a chemically modified M2 subtype. This suggests the presence of thiol groups controlling the antagonist binding activity of this muscarinic receptor subtype.
研究了用烷基化剂N-乙基马来酰亚胺（NEM）处理的牛气管平滑肌质膜部分中L-[二苯乙醇酸-4,4'-3H]喹核醇苯甲酸酯的结合情况。发现NEM（2.5 mM）可使最大结合量（Bmax）从1116 fmol/mg蛋白显著降低至853 fmol/mg蛋白，并使毒蕈碱型乙酰胆碱受体（mAchR）活性的解离常数（KD）值从36 pM增加至61 pM。使用选择性拮抗剂的竞争实验研究了这些质膜中的mAchR亚型。哌仑西平表现出低竞争活性，其抑制常数（pKi）为6.91±0.03，与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]苯二氮䓬-6-酮）相似（pKi = 6.90±0.04）；而六氢二苯哌啶（HDD）及其对氟衍生物（p-FHHSiD）显示出比哌仑西平更高的亲和力，其pKi值分别为7.45±0.05和7.17±0.06。拮抗剂4-二苯乙酰氧基-N-甲基哌啶甲基溴化物（4-DAMP）的pKi为8.25±0.03，与甲奥克明显示的亲和力无显著差异（pKi = 8.00±0.04）。这些数据表明，与从牛气道平滑肌分离的质膜部分相关的mAchR可归类为M2亚型毒蕈碱受体。NEM处理改变了mAchR对特定拮抗剂如甲奥克明的亲和力（Ki增加3倍），结果表明烷基化的mAchR表现为化学修饰的M2亚型。这表明存在控制该毒蕈碱受体亚型拮抗剂结合活性的巯基。
