Complete elucidation of mechanisms of actions of antitussives for developing novel cough-regulating drugs desired by aged peoples

在本项目中，我们首先利用全细胞和制氨抑素穿孔膜片钳技术研究了不同中枢作用的止咳药对单个神经元中5-HT_<1A>、GABA_B或肾上腺素α_2受体介导的不同电流的作用。其次，我们利用相同的技术研究了缓激肽（BK）对气管旁神经节（PTG）神经元的作用，以及BK是否会改变PTG神经元的毒蕈碱和烟碱反应。[结果]1)所有非麻醉性中枢作用的止咳药均抑制中缝背（DR）神经元5-HT_<1A>受体介导的内向电流（I_H<5-HT>）。这种作用可能是由于抑制了g蛋白偶联的与5-HT_<1A>受体偶联的内向矫正K^+通道（GIRK），在细胞内灌注GTP-γS的情况下，这些止咳剂有效地抑制了5-HT不可逆激活的GIRK电流。2) δ-拮抗剂，纳曲本（Nal）和纳曲多（naltrindole），具有止咳作用，对电流也有相同的作用。3) GABA_B受体激动剂Badofen也能激活DR神经元中的GIRK电流。4) DM、氯培斯汀（Clo）和Nal对GABA_B受体介导的电流也有浓度依赖性的抑制作用，IC_<50>s分别为8.23 × 10^<-6 >m、1.36 × 10^<-6 >m和4.36 × 10^<-6 >m。5) DM、do和Nal不仅抑制I_<5- ht >和I_<bac>。但同样浓度的α - 2肾上腺素受体介导的GIRK电流也存在。6) BK通过抑制m通道强烈激活PTG神经元。7)在PTG神经元中，BK对毒蕈碱反应有加性作用，对烟碱反应有协同作用。

In this project, at first, we investigated the actions of various centrally-acting antitussives on various currents mediated by 5-HT_<1A>, GABA_B or adrenergic α_2 receptors, in single brain neurons using whole cell and nystatin-perforated patch clamp techniques. Secondarily, we investigated the action of bradykinin (BK) on paratracheal ganglion (PTG) neurons, and further whether BK modified muscarinic and nicotinic responses in PTG neurons using the same techniques. [Results] 1) All non-narcotic centrally-acting antitussives studied inhibited 5-HT_<1A> receptor mediated inward current (I_H<5-HT>) in dorsal raphe (DR) neurons. This action was suggested to be due to inhibition of G-protein coupled inwardly rectifying K^+ channels (GIRK) coupled to 5-HT_<1A> receptor, because these antitussives effectively inhibited GIRK currents irreversibly activated by 5-HT under condition of intracellular perfusion of GTP-γS. 2) δ-Antagonists, naltriben (Nal) and naltrindole, which have an antitussive action, also have the same action on the currents. 3) Badofen, a GABA_B receptor agonist, also activated GIRK currents in DR neurons. 4) DM, cloperastine (Clo) and Nal also inhibited GABA_B receptor mediated currents in a concentration-dependent manner, with IC_<50>s of 8.23 × 10^<-6>M, 1.36 × 10^<-6>M and 4.36 × 10^<-6>M, respectively. 5) DM, do and Nal inhibited not only I_<5-HT> and I_<bac>. But also GIRK currents mediated by α2-adrenoceptor at almost the same concentration. 6) BK strongly activated PTG neurons through inhibition of M-channels. 7) In PTG neurons, BK caused additive effect on muscarinic responses and synergistic effect on nicotinic responses.