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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及其分型。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的钙荧光图象及细胞内游离钙离子浓度 ([Ca2 + ]i)的动态变化。结果 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 (10mmol/L)时引起部分 (4/ 5个 )离体VHC内 [Ca2 + ]i 升高 ;阿托品对ACh、CCh引起的VHC内 [Ca2 + ]i 的升高有抑制作用 ;加入 0 1mmol/L阿托品可使 1mmol/LAch或CCh引起的VHC内 [Ca2 + ]i 升高的峰值明显减小 (t检验均有显著差异P <0 0 1)。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。
Objective To investigate the effect of cholinergic receptor agonist on the intracellular calcium concentration in isolated vestibular hair cells to explore the cholinergic receptors and their types in the vestibular hair cell membrane. Methods The isolated vestibular hair cells (VHCs) of guinea pigs and the fluorescent probe 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 Both acetylcholine (ACh) and carbachol (CCh), both cholinergic and N-type receptor agonists, caused the increase of [Ca2 +] i in isolated VHC. The N-type receptor agonist, (AChBr) increased [Ca2 +] i in some (4/5) ex vivo VHCs only at high concentration (10 mmol / L); atropine induced a decrease in [Ca2 +] i (P <0.01). The addition of 0 1 mmol / L atropine significantly decreased the peak of [Ca2 +] i in VHC induced by 1 mmol / LAch or CCh. 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 caused by M-type receptor agonists.