Kitazawa T, Uchiyama F, Hirose K, Taneike T
Department of Pharmacology, Faculty of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, Japan.
Eur J Pharmacol. 1999 Feb 19;367(2-3):325-34. doi: 10.1016/s0014-2999(98)00946-7.
The aim of the present study was to characterize the subtype of muscarinic receptor that mediates acetylcholine-induced contractions in the nonpregnant proestrus swine myometrium by means of mechanical, radioligand ([3H]quinuclidinyl benzilate) binding and biochemical (measurement of cyclic AMP) approaches. Acetylcholine (-logEC50, 6.12), oxotremorine-methiodide (6.47), methacholine (6.35), carbachol (6.18) and muscarine (6.33) caused contractile responses of the uterine circular muscle, with a similar maximum amplitude, but pilocarpine and McN-A-343 (4-(m-chlorophenyl-carbamoyloxy)-2-butynyltrimethylammonium) were ineffective in causing contraction. The contractile response to acetylcholine was antagonized by the following muscarinic receptor antagonists in a competitive manner (with pA2 values in parentheses): atropine (8.95), 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 8.83), tropicamide (7.07), himbacine (7.01), pirenzepine (6.42) and 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyri do[2,3 b][1,4]benzodiazepin-6-one (AF-DX116, 5.96). Electrical field stimulation (10 Hz) caused tetrodotoxin- and atropine-sensitive contractions in the circular muscle. All muscarinic receptor antagonists decreased the electrical field stimulation-induced contraction in a concentration-dependent manner. The order of inhibition (-logIC50) was 4-DAMP (8.35) > tropicamide (6.72) > himbacine (6.54) > pirenzepine (6.31)> AF-DX116 (6.13). Acetylcholine did not affect the cytoplasmic cyclic AMP level, regardless of the presence or absence of forskolin, suggesting the absence of functional muscarinic M2 and/or M4 receptors in the swine myometrium. The receptor binding study indicated that circular muscle layers of the swine myometrium contained a single class of [3H]quinuclidinyl benzilate binding site (Kd = 0.92 nM; Bmax = 126.6 fmol/mg protein). Specific binding was displaced by muscarinic receptor antagonists in the following order (with pKi value and Hill coefficient in parentheses): atropine (8.22 and 0.93) > 4-DAMP (8.18 and 0.94) > tropicamide (6.78 and 0.93) > pirenzepine (5.46 and 0.92) > AF-DX116 (5.12 and 0.94). The present results suggest that in circular muscle layers of the swine myometrium, exogenous and endogenous acetylcholine cause contraction through activation of muscarinic M3 receptors present on smooth muscle cells.
本研究的目的是通过机械、放射性配体（[3H]喹核醇基苯甲酸酯）结合和生化（环磷酸腺苷测量）方法，鉴定介导乙酰胆碱诱导未孕动情前期猪子宫肌层收缩的毒蕈碱受体亚型。乙酰胆碱（-logEC50，6.12）、氧化震颤素甲碘化物（6.47）、醋甲胆碱（6.35）、卡巴胆碱（6.18）和毒蕈碱（6.33）引起子宫环行肌的收缩反应，最大幅度相似，但毛果芸香碱和 McN-A-343（4-（间氯苯基-氨甲酰氧基）-2-丁炔基三甲基铵）不能引起收缩。乙酰胆碱引起的收缩反应被下列毒蕈碱受体拮抗剂以竞争性方式拮抗（括号内为 pA2 值）：阿托品（8.95）、4-二苯乙酰氧基-N-甲基哌啶（4-DAMP，8.83）、托吡卡胺（7.07）、辛巴辛（7.01）、哌仑西平（6.42）和 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]苯并二氮杂卓-6-酮（AF-DX116，5.96）。电场刺激（10 Hz）引起环行肌中对河豚毒素和阿托品敏感的收缩。所有毒蕈碱受体拮抗剂均以浓度依赖性方式降低电场刺激诱导的收缩。抑制顺序（-logIC50）为 4-DAMP（8.35）>托吡卡胺（6.72）>辛巴辛（6.54）>哌仑西平（6.31）>AF-DX116（6.13）。无论有无福司可林，乙酰胆碱均不影响细胞质环磷酸腺苷水平，提示猪子宫肌层中不存在功能性毒蕈碱 M2 和/或 M4 受体。受体结合研究表明，猪子宫肌层的环行肌层含有一类单一的[3H]喹核醇基苯甲酸酯结合位点（Kd = 0.92 nM；Bmax = 126.6 fmol/mg 蛋白质）。特异性结合被毒蕈碱受体拮抗剂以下列顺序取代（括号内为 pKi 值和希尔系数）：阿托品（8.22 和 0.93）>4-DAMP（8.18 和 0.94）>托吡卡胺（6.78 和 0.93）>哌仑西平（5.46 和 0.92）>AF-DX116（5.12 和 0.94）。目前的结果表明，在猪子宫肌层的环行肌层中，外源性和内源性乙酰胆碱通过激活平滑肌细胞上存在的毒蕈碱 M3 受体引起收缩。
