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目的 通过观察胆碱能受体激动剂对离体前庭毛细胞内钙离子浓度的影响 ,以探讨前庭毛细胞膜所存在的胆碱能受体分型及庆大霉素对钙离子通道的阻断作用。 方法 用胶原酶消化后机械分离法 ,分离豚鼠前庭毛细胞 (VHC) ,钙敏荧光探针 Fluo- 3染色 ,用激光扫描共聚焦显微镜记录 VHC的钙荧光图像及细胞内游离钙离子浓度 ([Ca2 + ]i)的动态变化。结果 1胆碱能 M和 N型受体激动剂乙酰胆碱 (ACh)、氨甲酰胆碱 (CCh)均可引起离体 VHC内 [Ca2 + ]i 的升高 ;N型受体激动剂溴化乙酰胆碱 (ACh- Br)仅在高浓度 (10 mm ol/ L )时引起部分 (4 / 5个 )离体 VHC内 [Ca2 + ]i 升高 ,在低浓度 (1mm ol/ L)时影响不明显 ;2阿托品对 ACh、CCh引起的 VHC内 [Ca2 + ]i 的升高有抑制作用 ;加入 0 .1mm ol/ L阿托品可使 1m mol/ L ACh或 CCh引起的 VHC内 [Ca2 + ]i 升高的峰值明显减小 (P<0 .0 1) ;3 0 .1m mol/ L ACh引起 VHC内 [Ca2 + ]i 升高之后 ,再加入 0 .5 m mol/ L 庆大霉素 ,VHC内 [Ca2 + ]i 出现显著下降 ,至低于静息时的水平。 结论 豚鼠壶腹嵴前庭毛细胞膜上存在 M型和 N型两种受体 ,M型受体激动剂引起 VHC内 [Ca2 + ]i 的升高较 N型明显。阿托品对 M型受体激动剂引起的 [Ca2 + ]i升高有抑制作用 ;庆大霉素对前庭
Objective To investigate the effect of cholinergic receptor agonist on the intracellular calcium concentration in isolated vestibular hair cells in order to investigate the cholinergic receptor type in vestibular hair cell membrane and the blocking of calcium channel by gentamicin effect. Methods The isolated vestibular hair cells (VHCs) and Fluorescein-sensitive fluorescent probes (Fluo-3) were isolated by mechanical separation after collagenase digestion. The calcium fluorescence images and intracellular free calcium concentration of VHC were recorded by laser scanning confocal microscopy ([ Ca2 +] i) dynamic changes. Results Choline M and N-type receptor agonists, acetylcholine (ACh) and carbachol (CCh), all induced the increase of [Ca2 +] i in isolated VHC; N-type receptor agonist Acetylcholine (ACh-Br) caused a partial (4/5) increase in [Ca2 +] i in ex vivo VHC only at high concentrations (10 mmoles / L) and at low concentrations 2 atropine inhibited the increase of [Ca2 +] i in VHC induced by ACh and CCh. Adding 0.1mol / L atropine could make [Ca2 +] i in VHC induced by 1m mol / L ACh or CCh (P <0.01). After the increase of [Ca2 +] i in VHC induced by 30 mol / L ACh, 0.5 mmol / L gentamycin was added, There was a significant decrease in [Ca2 +] i in VHC, below the level at rest. Conclusion There are M and N type receptors on membrane of vestibular epithelium of guinea pig ampulla. M type receptor agonist induced the increase of [Ca2 +] i in VHC compared with N type. Atropine inhibits the increase of [Ca2 +] i induced by M-type receptor agonist;