水杨酸钠对豚鼠耳蜗单离外毛细胞游离钙浓度影响及其调控机制研究

Sodium salicylate induced intracellular Ca^(2+) concentration change in guinea pig outer hair cells

目的 观察水杨酸钠对豚鼠耳蜗外毛细胞 (outerhaircells,OHC)内游离钙浓度 [Ca2 + ]i的影响 ,并对钙通道调控药物的拮抗作用进行观察 ,以深入探讨水杨酸钠耳毒性的分子生物学机制。方法 豚鼠全身麻醉后断头活杀 ,酶 机械法分离获得耳蜗单离OHC。 10 μmol/L的Fluo 3/AM负载30min后用ACASUltima型激光扫描共聚焦显微镜观察不同药物灌流下OHC[Ca2 + ]i的变化。结果 OHC在标准细胞外液灌流过程中 [Ca2 + ]i保持稳定。 1mmol/L水杨酸钠灌流致 8/ 10个细胞的 [Ca2 + ]i明显升高。无钙细胞外液中灌流亦有 10 / 10个细胞 [Ca2 + ]i明显的升高。经 3mmol/L利多卡因作用1h后 ,水杨酸钠灌流不能使细胞 [Ca2 + ]i升高 (n =10 )。盐酸氟桂嗪可以明显阻断水杨酸钠导致的OHC[Ca2 + ]i升高效应 (3/ 12细胞的 [Ca2 + ]i升高 ) ,而尼莫地平无此效应 (7/ 7细胞的 [Ca2 + ]i升高 )。结论 水杨酸钠可以导致OHC[Ca2 + ]i明显增加 ,OHC[Ca2 + ]i升高来源于细胞内钙库的动员和释放。使细胞内 [Ca2 + ]i升高可能是水杨酸钠的耳毒性机制之一。

Objective In order to explore the mechanism of sodium salicylate ototoxicity the effect of sodium salicylate on intracellular Ca 2+ ([Ca 2+ ]i ) of the outer hair cells (OHCs) and the effects of calcium channel antagonist on sodium salicylate-induced [Ca 2+ ]i changes of the OHCs were examined Methods The OHCs of guinea pig cochlear were isolated using an enzyme-machine methods and loaded with 10 μmol/L Fluo-3/AM for 30 min at 37 ℃ Individual Fluo-3 loaded OHC was examined with a confocal microscope (ACAS Ultima, USA) using a 20 x objective lens and linear scan mean The fluorescent images, collected each 5-sec for 300 sec, were stored in a computer The fluorescent intensity of the OHCs were analyzed by the software cooperated with the confocal microscope, and a curve of fluorescent intensity changes trend against time was obtained Results The [Ca 2+ ]i of OHCs were steady under normal extracellular liquid perfusion Salicylate increased [Ca 2+ ]i in OHCs in calcium-free medium(10/10) and standard medium (8/10) 3 mmol/L lidocaine inhibited [Ca 2+ ]i increase in OHCs induced by salicylate(0/10) The [Ca 2+ ]i increase in OHCs induced by salicylate could be blocked by flunarizine(3/12) Nimodipin failed to block the [Ca 2+ ]i increase of OHCs induced by salicylate (7/7) Conclusion Sodium salicylate can result in [Ca 2+ ]i increase in OHC markedly,which may be due to the Ca 2+ release from intracellular calcium stores The effects of salicylate on[Ca 2+ ]i in OHCs might be one of the mechanisms of salicylate ototoxicity