豚鼠耳蜗单离外毛细胞游离钙的胆碱能调控

Cholinergic Regulation of Intracellular Free Calcium Concentrations in the Isolated Outer Hair Cells of the Guinea Pig Cochlea

目的 观察胆碱能兴奋剂和拮抗剂对豚鼠耳蜗单离外毛细胞 (OHC)内游离钙浓度 ([Ca2 + ]i)的影响 ,探讨OHC内 [Ca2 + ]i的胆碱能调控机制。方法 健康豚鼠 15只处死 ,Hanks液中分离活性单离OHC ,Fluo - 3/AM负载 30min。分 7组进行不同干预 ,包括乙酰胆碱 (ACH ,胆碱能M和N受体兴奋剂 )、毒蕈碱 (M受体兴奋剂 )、阿托品 (M受体拮抗剂 )、庆大霉素 (N受体拮抗剂 )、加Hanks液对照组和不加干预对照组 ;另 1组为dHanks液中加ACh。用激光扫描共聚焦显微镜 (Bio-Rad ,英国 )观察OHC内 [Ca2 + ]i的变化。结果 Hanks液中不加干预试剂、加毒蕈碱、加Hanks液对照和无钙dHanks液中加ACh等 4组 ,[Ca2 + ]i在 2 5～ 30min内升幅 <1.10倍。Hanks液中加ACh组 [Ca2 + ]i最高升幅平均为 3.0 9倍 ;阿托品 +ACh组 [Ca2 + ]i为 2 .92倍 ;庆大霉素 +ACh组[Ca2 + ]i为 1.81倍。结论 豚鼠耳蜗传出神经递质ACh通过兴奋N受体 ,开放OHC的细胞膜钙离子通道 ,使细胞外Ca2 + 内流外毛细胞内 ;[Ca2 + ]i增加需要细胞外液中钙离子存在。

Objective To observe the effects of cholinergic receptor agonists and antagonists on intracellular free Ca 2+ concentrations ([Ca 2+ ]i) of outer hair cells (OHCs) and to explore the mechanism of cholinergic regulation on [Ca 2+ ]i of OHCs. Methods After the guinea pigs were killed by decapitation, the bullae were removed and opened. Then the OHCs were isolated and loaded with 5 μmol Fluo-3/AM in Hanks' balanced salt solution (HBSS) for 30 min at room temperature. Fluo-3/AM loaded OHCs were excited at 488 nm line of an Ar-Kr laser using Bio-Rad MRC-1024 Laser Scan Confocal Microscpe (Bio-Rad, UK) for 25～30 min after treatment by nothing, acetylcholine (ACh, 1 mmol), gentamicin (10 mmol) + ACh, atropine (10 μmol) + ACh, HBSS as control, ACh added to OHCs bothed in dHBSS (without calcium), respectively. OHCs fluorescence were monitored every 10 sec for 25～30 min and the images were recorded. Results [Ca 2+ ]i of OHCs were slowly increased to ≤1.10 folds of their basic levels when 3 OHCs bathed in HBSS treated with nothing, 3 OHCs with HBSS as control, 6 OHCs with 1mmol muscarine, or 3 OHCs in dHBSS treated with 1mmol ACh, in 25-30 minutes respectively. The average peak of [Ca 2+ ]i was increased to 3.09 folds in 6 OHCs which were bathed in HBSS with 1mmol ACh, 2.92 folds in 6 OHCs in HBSS with 10μmol atropine + 1 mmol ACh, 1.81 folds in 6 OHCs in HBSS with 10 mmol gentamicin + 1 mmol ACh, respectively. Conclusion The data indicate that ACh, the neurotransmitter of efferent auditory nerve in cochlea, activates the nicotinic receptor to open the calcium channel in OHC membrane and increase [Ca 2+ ]i of OHCs, which is extracellular Ca 2+ -dependent.