Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle
Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle
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DOI：
10.1007/s00210-007-0223-1
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发表时间：
2008-06-01
影响因子：
3.6
通讯作者：
Taneike, Tetsuro
中科院分区：
文献类型：
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作者：
Kitazawa, Takio;Hirama, Ryuichi;Taneike, Tetsuro
使用对照（DDY和野生型）小鼠、毒蕈碱M-2或M-3单受体敲除（M2 KO、M3 KO）以及M-2和M-3受体双敲除小鼠（M-2/M3 KO），通过药理学和分子生物学研究对小鼠子宫中存在的功能性毒蕈碱乙酰胆碱受体进行了表征。卡巴胆碱（10 nM-100 μ M）以浓度依赖性方式增加对照小鼠子宫肌条的肌张力和阶段性收缩活动。卡巴胆碱诱导的最大收缩（E-max）在子宫的宫颈和卵巢区域之间不同。发情周期的阶段没有显着影响卡巴胆碱浓度-反应关系。河豚毒素不能减少卡巴胆碱引起的收缩，但毒蕈碱受体拮抗剂（11-[[2-[（二乙基氨基甲基）-1-哌啶基]乙酰基]-5，11-二氢-6H-吡啶并[2，3-B][2，3-B][1，4]苯并二氮杂卓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-11 H-吡啶并[2，3-B][1，4]苯并二氮杂卓-11-甲酰胺（AF-DX 384）、4-二苯基乙酰氧基-N-甲基-哌啶（4-DAMP）、对氟-六氢-硅-地芬尼多（p-F-HHSiD）、喜巴辛、甲氧曲明、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pK（B）值与这些M-3毒蕈碱受体拮抗剂的已知pK（i）值相关良好。在用百日咳毒素（100 μ g/kg，腹腔注射96小时）处理的小鼠的子宫条中，卡巴胆碱的E-max值显著降低，但引起50% E-max值的有效浓度（EC 50）保持不变。在用4-DAMP芥子气（30 nM）和AF-DX116（1 μ M）处理的子宫条中，随后用AF-DX116洗脱，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（氧代震颤素-M）均不引起任何收缩反应。通过逆转录聚合酶链反应在小鼠子宫中检测到M-2和M-3毒蕈碱受体信使RNA。卡巴胆碱也引起从M2 KO小鼠分离的子宫条收缩，但浓度-反应曲线与相应的野生型小鼠相比向右和向下移动。另一方面，从M3 KO和M-2/M-3双KO小鼠分离的子宫条几乎对卡巴胆碱不敏感。总之，虽然M-2和M-3毒蕈碱受体在小鼠子宫中表达，卡巴胆碱诱导的收缩反应主要由M-3受体介导。单独激活M-2受体不会引起子宫收缩;然而，M-2受体激活增强了小鼠子宫中M-3受体介导的收缩。
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 M-2 or M-3 single receptor knockout (M2KO, M3KO), and M-2 and M-3 receptor double knockout mice (M-2/M3KO). Carbachol (10 nM-100 mu M) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (E-max) 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 pK(b) values for these muscarinic receptor antagonists correlated well with the known pK(i) values of these antagonists for the M-3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 mu g/kg, i.p. for 96 h), E-max values for carbachol were significantly decreased, but effective concentration that caused 50% of E-max values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 mu M), 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 M-2 and M-3 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 M-2/M-3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M-2 and M-3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M-3 receptor. Activation of M-2 receptors alone did not cause uterine contractions; however, M-2 receptor activation enhanced M-3 receptor-mediated contractions in the mouse uterus.