Kitazawa Takio, Asakawa Koichi, Nakamura Tatsuro, Teraoka Hiroki, Unno Toshihiro, Komori Sei-Ichi, Yamada Masahisa, Wess Jürgen
Department of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069-8501, Japan.
J Pharmacol Exp Ther. 2009 Aug;330(2):487-93. doi: 10.1124/jpet.109.153304. Epub 2009 May 8.
The potential functional roles of M(3) muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M(2) or M(3) receptor single knockout (M(2)KO, M(3)KO), and M(2) and M(3) muscarinic receptor double knockout mice (M(2)/M(3)KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M(2) receptor mRNA was expressed predominantly in mouse atria but that the M(1), M(3), M(4), and M(5) receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 microM) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M(2)KO mice confirmed that this activity is mediated by the M(2) receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M(3)KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M(2)KO mice, carbachol showed only positive inotropic activity. In atria from M(2)/M(3) double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M(3) muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M(2) receptor activation on atrial contractility.
运用药理学和分子生物学技术，以野生型小鼠、毒蕈碱M(2)或M(3)受体单敲除小鼠（M(2)KO、M(3)KO）以及M(2)和M(3)毒蕈碱受体双敲除小鼠（M(2)/M(3)KO）为实验对象，研究了M(3)毒蕈碱受体在小鼠心房中的潜在功能作用。实时定量逆转录聚合酶链反应分析表明，M(2)受体mRNA在小鼠心房中表达占主导地位，但M(1)、M(3)、M(4)和M(5)受体亚型也有低水平表达。卡巴胆碱（10 nM - 30 μM）降低了从野生型小鼠分离的右心房的自发搏动频率。使用亚型选择性拮抗剂对M(2)KO小鼠心房进行的研究证实，该活性由M(2)受体亚型介导。在野生型小鼠的左心房中，卡巴胆碱降低了电场刺激诱发的收缩幅度（负性肌力作用），但这种抑制是短暂的，随后收缩幅度逐渐增加（正性肌力反应）。在M(3)KO小鼠的心房中，卡巴胆碱的短暂负性肌力作用转变为持续的负性肌力作用。相反，在M(2)KO小鼠的心房中，卡巴胆碱仅表现出正性肌力活性。在M(2)/M(3)双敲除小鼠的心房中，卡巴胆碱没有任何肌力活性。这些观察结果，辅以使用亚型选择性拮抗剂的功能研究，令人信服地证明心房M(3)毒蕈碱受体介导小鼠心房的正性肌力作用。从生理角度来看，这种活性可能有助于减弱M(2)受体激活对心房收缩力的抑制作用。
