Guo Chang-Kai, Zhang Song, Kong Wei-Jia, Li Qing-Tian, Li Zhi-Wang
Department of Otolaryngology, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Sheng Li Xue Bao. 2006 Apr 25;58(2):157-63.
Molecular biological studies and electrophysiological data have demonstrated that acetylcholine (ACh) is the principal cochlear and vestibular efferent neurotransmitter among mammalians. However, the functional roles of ACh in type II vestibular hair cells among mammalians are still unclear, with the exception of the well-known alpha9-containing nicotinic ACh receptor (alpha9-nAChR) in cochlear hair cells and frog saccular hair cells. In this study, the properties of the ACh-sensitive current were investigated by whole-cell patch clamp technique in isolated type II vestibular hair cells of guinea pigs. The direct effect of extracellular ACh was to induce a hyperpolarization effect in type II vestibular hair cells. Type II vestibular hair cells displayed a sustained outward current in response to the perfusion of ACh. It took about 60 s for the ACh-sensitive current to get a complete re-activation. The reversal potential of the ACh-sensitive current was (-66 +/- 8) mV, which indicated that potassium ion was the main carrier of this current. The blocking effect by the submillimolar concentration of tetraethylammonium (TEA) further indicated that extracellular ACh stimulated the calcium-dependent potassium current. Following replacement of the compartment of NaCl in the normal external solution with TrisCl, LiCl or saccharose respectively, the amplitude of the ACh-sensitive current was not affected. Blocking of the release of intracellular Ca(2+) stores by intracellular application of heparin failed to inhibit the ACh-sensitive current. Therefore, extracellular Na(+)and the inositol 1,4,5-trisphosphate (IP(3))-dependent intracellular Ca(2+)release were not involved in the activation of the ACh-sensitive current. However, the ACh-sensitive current was strongly affected by the concentration of the extracellular K(+), extracellular Ca(2+) and intracellular Mg(2+). The amplitude of the ACh- sensitive current was strongly inhibited by high concentration of extracellular K(+). In the Ca(2+)-free external solution, ACh only activated a very small current; however, the ACh-sensitive current demonstrated a Ca(2+)-dependent inhibition effect in high concentration of Ca(2+)solution. In addition, the ACh-sensitive current was inhibited by increasing of the concentration of intracellular Mg(2+). In conclusion, the present results demonstrate that ACh plays an important role in the vestibular efferent system. The fact that Na(+) is not involved in the ACh-sensitive current will not favor the well-known profile of alpha9-nAChR, which is reported to display a small but important permeability to Na(+). It is also suggested that, in vivo, the amplitude of the ACh-induced hyperpolarization may strongly depend on the concentration of extracellular Ca(2+)and intracellular Mg(2+).
分子生物学研究和电生理数据表明，乙酰胆碱（ACh）是哺乳动物耳蜗和前庭传出神经的主要神经递质。然而，除了耳蜗毛细胞和青蛙球囊毛细胞中众所周知的含α9的烟碱型乙酰胆碱受体（α9-nAChR）外，ACh在哺乳动物II型前庭毛细胞中的功能作用仍不清楚。在本研究中，采用全细胞膜片钳技术，在豚鼠分离的II型前庭毛细胞中研究了ACh敏感电流的特性。细胞外ACh的直接作用是在II型前庭毛细胞中诱导超极化效应。II型前庭毛细胞在灌注ACh时表现出持续的外向电流。ACh敏感电流大约需要60秒才能完全重新激活。ACh敏感电流的反转电位为（-66±8）mV，这表明钾离子是该电流的主要载体。亚毫摩尔浓度的四乙铵（TEA）的阻断作用进一步表明，细胞外ACh刺激了钙依赖性钾电流。分别用TrisCl、LiCl或蔗糖替代正常外部溶液中的NaCl区室后，ACh敏感电流的幅度不受影响。通过细胞内应用肝素阻断细胞内Ca（2+）储存的释放未能抑制ACh敏感电流。因此，细胞外Na（+）和肌醇1,4,5-三磷酸（IP（3））依赖性细胞内Ca（2+）释放不参与ACh敏感电流的激活。然而，ACh敏感电流受到细胞外K（+）、细胞外Ca（2+）和细胞内Mg（2+）浓度的强烈影响。高浓度的细胞外K（+）强烈抑制ACh敏感电流的幅度。在无Ca（2+）的外部溶液中，ACh仅激活非常小的电流；然而，在高浓度的Ca（2+）溶液中，ACh敏感电流表现出Ca（2+）依赖性抑制作用。此外，ACh敏感电流受到细胞内Mg（2+）浓度增加的抑制。总之，目前的结果表明，ACh在前庭传出系统中起重要作用。Na（+）不参与ACh敏感电流这一事实与众所周知的α9-nAChR的特征不符，据报道α9-nAChR对Na（+）具有小但重要的通透性。还表明，在体内，ACh诱导的超极化幅度可能强烈依赖于细胞外Ca（2+）和细胞内Mg（2+）的浓度。
