Kitazawa Takio, Hirama Ryuichi, Masunaga Kozue, Nakamura Tatsuro, Asakawa Koichi, Cao Jinshan, Teraoka Hiroki, Unno Toshihiro, Komori Sei-ichi, Yamada Masahisa, Wess Jürgen, Taneike Tetsuro
Department of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido, 069-8501, Japan.
Naunyn Schmiedebergs Arch Pharmacol. 2008 Jun;377(4-6):503-13. doi: 10.1007/s00210-007-0223-1. Epub 2007 Dec 11.
Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via reverse transcription polymerase chain reaction. Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the concentration-response curve was shifted to the right and downward compared with that for the corresponding wild-type mice. On the other hand, uterine strips isolated from M3KO and M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor. Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor activation enhanced M3 receptor-mediated contractions in the mouse uterus.
通过药理学和分子生物学研究，利用对照（DDY和野生型）小鼠、毒蕈碱M2或M3单受体敲除小鼠（M2KO、M3KO）以及M2和M3受体双敲除小鼠（M2/M3KO），对存在于小鼠子宫中的功能性毒蕈碱型乙酰胆碱受体进行了表征。卡巴胆碱（10 nM - 100 μM）以浓度依赖性方式增加对照小鼠子宫条带的肌张力和相性收缩活性。子宫颈和卵巢区域的子宫条带中，卡巴胆碱诱导的最大收缩（Emax）有所不同。发情周期阶段对卡巴胆碱浓度 - 反应关系无显著影响。河豚毒素不会降低卡巴胆碱诱导的收缩，但毒蕈碱受体拮抗剂（11 - [[2 - [(二乙氨基甲基)-1 - 哌啶基]乙酰]-5,11 - 二氢 - 6H - 吡啶并[2,3 - b][2,3 - b][1,4]苯并二氮杂䓬 - 6 - 酮（AF - DX116）、N - [2 - [2 - [(二丙氨基甲基)-1 - 哌啶基]乙基]-5,6 - 二氢 - 6 - 氧代 - 11H - 吡啶并[2,3 - b][1,4]苯并二氮杂䓬 - 11 - 甲酰胺（AF - DX384）、4 - 二苯基乙酰氧基 - N - 甲基 - 哌啶（4 - DAMP）、对氟 - 六氢 - 硅 - 二苯地洛（p - F - HHSiD）、辛巴辛、甲奥克明、哌仑西平和托吡卡胺）以竞争性方式抑制卡巴胆碱诱导的收缩。这些毒蕈碱受体拮抗剂的pKb值与这些拮抗剂对M3毒蕈碱受体的已知pKi值相关性良好。在从用百日咳毒素（100 μg/kg，腹腔注射96小时）处理的小鼠分离的子宫条带中，卡巴胆碱的Emax值显著降低，但引起50% Emax值的有效浓度（EC50）保持不变。在用4 - DAMP芥子碱（30 nM）和AF - DX116（1 μM）处理子宫条带，随后洗脱AF - DX116后，卡巴胆碱和N,N,N - 三甲基 - 4 - (2 - 氧代 - 1 - 吡咯烷基)-2 - 丁炔 - 1 - 碘化铵（氧化震颤素 - M）均未引起任何收缩反应。通过逆转录聚合酶链反应在小鼠子宫中检测到M2和M3毒蕈碱受体信使核糖核酸。卡巴胆碱也引起从M2KO小鼠分离的子宫条带收缩，但与相应野生型小鼠相比，浓度 - 反应曲线向右下方移动。另一方面，从M3KO和M2/M3双敲除小鼠分离的子宫条带对卡巴胆碱几乎不敏感。总之，虽然M2和M3毒蕈碱受体均在小鼠子宫中表达，但卡巴胆碱诱导的收缩反应主要由M3受体介导。单独激活M2受体不会引起子宫收缩；然而，M2受体激活增强了小鼠子宫中M3受体介导的收缩。
