豚鼠壶腹嵴毛细胞内钙离子活动及ATP的影响

目的 探讨离体前庭毛细胞（ＶＨＣ）去极化时细胞内钙离子活动特征及内耳活性物质ＡＴＰ对离体ＶＨＣ内钙离子浓度的影响及其机制。方法 采用酶孵育后机械分离法分离豚鼠壶腹嵴ＶＨＣ，６μｍｏｌ／Ｌ钙荧光探针Ｆｌｕｏ－３染色，利用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下、加入高Ｋ＾＋液及不同浓度ＡＴＰ时代表ＶＨＣ内［Ｃａ＾２＋ｉ的荧光强度相对值的变化。结果 细胞外有Ｃａ＾２＋存在时，７５ｎｍｏｌ／ＬＫ＾

Intracellular calcium mobilization and effects of ATP on ampullate crista hair cells of the guinea pig]

OBJECTIVE: To further understand the intracellular calcium mobilization features of the isolated ampullate crista vestibular hair cells(VHCs) and ATP effects on intracellular free calcium concentration([Ca2+]i) in VHCs of the guinea pig. METHODS: Vestibular hair cells were isolated from the guinea pig crista ampullaris by enzymatic and mechanical methods. The influence of high K+ solution and ATP on [Ca2+]i was investigated. The [Ca2+]i of VHC was examined using laser scanning confocal microscopy(LSCM) and the Ca2+ sensitive dye Fluo-3. RESULTS: (1) 75 mmol/L K+ Hank's solution resulted in an increase of [Ca2+]i in both the cytoplasm and the cuticular plate of 5 type I VHCs. In the Ca(2+)-free medium, however, 75 mmol/L K+ solution could not induce [Ca2+]i an increase in observed 5 type I VHCs. (2) In the presence of 1.0 mmol/L or 0.1 mmol/L of ATP, there was a rapid reversible rise of the [Ca2+]i in 18/19 type I VHCs. A small response of ATP-induced [Ca2+]i was found only in 1/10 of type I VHC in Ca(2+)-free medium. There was no significant increase in [Ca2+]i when the same volume of Hank's solution was applied to the chamber containing VHCs. CONCLUSIONS: The result suggests that high K+ solution led to Ca2+ influx via voltage-dependent calcium channels. Extracellular Ca2+ influx is a major source of the ATP-induced [Ca2+]i increase.