阳离子对豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的调制

本文旨在探讨哺乳动物前庭胆碱能传出神经系统的作用机制,应用全细胞膜片钳技术研究新鲜分离的豚鼠Ⅱ型前庭毛细胞ACh-敏感性电流的特性以及细胞内外的阳离子对ACh-敏感性电流的调制作用。结果显示,Ⅱ型前庭毛细胞对细胞外ACh敏感,ACh激活缓慢持久的外向性电流,室温下此电流的再次完全激活时间约为(60±10)s。ACh-敏感性电流的反转电位为(-66±8)mV,提示此电流主要由K+参与形成,其直接作用是使毛细胞超极化。ACh-敏感性电流对较高浓度的四乙胺(tetraethylammonium chloride,TEA)敏感,提示细胞外ACh激活钙依赖性钾电流。进一步检测细胞内外阳离子对 ACh-敏感性电流的调制作用发现,细胞外Na+和细胞内Ca2+释放不参与此电流的激活过程,而细胞外K+、细胞外Ca2+和细胞内Mg2+对ACh-敏感性电流具有重要的调制作用。进一步提示,ACh是哺乳动物前庭传出神经系统重要的神经递质。 Na+不参与ACh-敏感性电流的激活过程提示,ACh-敏感性电流可能由非α9-N型胆碱能受体(α9-nAChR)介导。ACh诱导的Ⅱ型前庭毛细胞超极化作用受细胞外Ca2+浓度和细胞内Mg2+浓度调制。

Cation ions modulate the ACh-sensitive current in type II vestibular hair cells of guinea pigs

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+).