Unno Toshihiro, Matsuyama Hayato, Sakamoto Takashi, Uchiyama Mai, Izumi Yusuke, Okamoto Hiroyuki, Yamada Masahisa, Wess Jürgen, Komori Seiichi
Laboratory of Pharmacology, Department of Veterinary Medicine, Faculty of Applied Biological Science, Gifu University, Japan.
Br J Pharmacol. 2005 Sep;146(1):98-108. doi: 10.1038/sj.bjp.0706300.
Isometric contractile responses to carbachol were studied in ileal longitudinal smooth muscle strips from wild-type mice and mice genetically lacking M(2) or M(3) muscarinic receptors, in order to characterize the mechanisms involved in M(2) and M(3) receptor-mediated contractile responses. Single applications of carbachol (0.1-100 microM) produced concentration-dependent contractions in preparations from M(2)-knockout (KO) and M(3)-KO mice, mediated via M(3) and M(2) receptors, respectively, as judged by the sensitivity of contractile responses to blockade by the M(2)-preferring antagonist methoctramine (300 nM) or the M(3)-preferring antagonist 4-DAMP (30 nM). The M(2)-mediated contractions were mimicked in shape by submaximal stimulation with high K(+) concentrations (up to 35 mM), almost abolished by voltage-dependent Ca(2+) channel (VDCC) antagonists or depolarization with 140 mM K(+) medium, and greatly reduced by pertussis toxin (PTX) treatment. The M(3)-mediated contractions were only partially inhibited by VDCC antagonists or 140 mM K(+)-depolarization medium, and remained unaffected by PTX treatment. The contractions observed during high K(+) depolarization consisted of different components, either sensitive or insensitive to extracellular Ca(2+). The carbachol contractions observed with wild-type preparations consisted of PTX-sensitive and -insensitive components. The PTX-sensitive component was functionally significant only at low carbachol concentrations. The results suggest that the M(2) receptor, through PTX-sensitive mechanisms, induces ileal contractions that depend on voltage-dependent Ca(2+) entry, especially associated with action potential discharge, and that the M(3) receptor, through PTX-insensitive mechanisms, induces contractions that depend on voltage-dependent and -independent Ca(2+) entry and intracellular Ca(2+) release. In intact tissues coexpressing M(2) and M(3) receptors, M(2) receptor activity appears functionally relevant only when fractional receptor occupation is relatively small.
为了阐明M(2)和M(3)受体介导的收缩反应所涉及的机制，研究了野生型小鼠以及基因敲除M(2)或M(3)毒蕈碱受体的小鼠回肠纵行平滑肌条对卡巴胆碱的等长收缩反应。单次应用卡巴胆碱（0.1-100 microM）可使M(2)基因敲除（KO）和M(3)基因敲除小鼠的标本产生浓度依赖性收缩，分别通过M(3)和M(2)受体介导，这可根据收缩反应对M(2)选择性拮抗剂美索曲明（300 nM）或M(3)选择性拮抗剂4-DAMP（30 nM）阻断的敏感性来判断。用高浓度K(+)（高达35 mM）进行次最大刺激可模拟M(2)介导的收缩形状，电压依赖性Ca(2+)通道（VDCC）拮抗剂或用140 mM K(+)培养基进行去极化几乎可消除该收缩，百日咳毒素（PTX）处理可使其大大降低。M(3)介导的收缩仅部分被VDCC拮抗剂或140 mM K(+)去极化培养基抑制，且不受PTX处理的影响。在高K(+)去极化期间观察到的收缩由对细胞外Ca(2+)敏感或不敏感的不同成分组成。野生型标本中观察到的卡巴胆碱收缩由PTX敏感和不敏感成分组成。PTX敏感成分仅在低卡巴胆碱浓度时具有功能意义。结果表明，M(2)受体通过PTX敏感机制诱导依赖于电压依赖性Ca(2+)内流的回肠收缩，尤其是与动作电位发放相关的收缩，而M(3)受体通过PTX不敏感机制诱导依赖于电压依赖性和非依赖性Ca(2+)内流以及细胞内Ca(2+)释放的收缩。在共表达M(2)和M(3)受体的完整组织中，仅当受体占有率相对较小时，M(2)受体活性在功能上才显得重要。
