新生大鼠耳蜗血管纹缘细胞释放ATP的机制

Mechanism of ATP release from cultured marginal cells of stria vascularis in neonatal rat

目的证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP，并进一步探讨缘细胞释放ATP的机制。方法分离、培养新生大鼠耳蜗血管纹缘细胞，采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯（didecyladipate，DDA）、细胞外K＋、毒胡萝卜素、细胞外Ca^2＋、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响。结果随着巴佛洛霉素A1浓度的增加，细胞外液中ATP的浓度明显下降；当DDA浓度增加时，细胞外液中ATP的浓度几乎呈线性增加。随着细胞外液中的K＋浓度的增高，缘细胞释放的ATP浓度呈现上升趋势，当细胞外液中的K＋浓度为9．15mmol／L时，ATP的释放量达到峰值，之后随着K＋浓度的继续升高ATP的释放量呈下降趋势。随着毒胡萝卜素浓度的增加，缘细胞释放的ATP浓度呈现明显下降的趋势。当细胞外Ca^2＋浓度为0mmol／L时，缘细胞仍然释放ATP；Ca^2＋浓度增加与ATP的释放呈负相关，但当细胞外的Ca“浓度达到1．25mmol／L以上时，ATP的释放量维持在一个较稳定的水平。U73122的浓度在0．25-1．25μmol／L时，其与缘细胞释放的ATP浓度呈负相关。当马兜铃酸钠的浓度为12．5-100．0ixmol／L，缘细胞ATP释放呈明显下降的趋势；当其浓度〉100．0μmol／L时，ATP释放浓度趋于平稳。结论体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP，其释放量与钙泵、K＋通道状态以及细胞内信号传导通路相关酶的活性有关。

Objective To further confirm release of adenosine triphosphate （ATP） from cultured marginal cells in vitro of stria vascularis in neonatal rat, and to explore the mechanism of ATP release from marginal cells. Methods Isolation and in vitro culture of marginal cells of neonatal rats＇ cochlea. ATP released by marginal cells in extracellular fluid were detected using biolumineseence assay when add regants separately as follow： bafilomycin A1, didecyl adipate （DDA） , extracellular K ＋ , thapsigargin, extracellular Ca2＋, U73122, and aristolochic acid. Results The concentrations of ATP in the extracellular fluid significantly and gradually decreased along with increasing concentrations of bafilomycin A1. The concentrations of ATP in the extracellular fluid were in linear increased with DDA was added to marginal cellsuspensions. ATP concentrations increased as the concentration ot extracellular K＋ was increased, and reached the peak with a K＋ concentration of 9.15 mmol/L. At higher K＋ concentrations, ATP concentrations decreased. With the addition of increasing concentrations of thapsigargin to test marginal cells, ATP concentrations were significantly decreased. When extracellular Ca^2＋ was completely chelated, marginal cells continued to release ATP. Moreover, as extracellular Ca^2＋ increased, the release of ATP decreased. However, the amount of ATP releas remained to a baseline when extracellular concentration of Ca2＋ reached 1.25 mmol/L or above. When concentrations of U73122 remained within the range of 0. 25 to 1.25μmol/L, as U73122 increased, the release of ATP decreased. When concentrations of aristolochic acid ranging from 12. 5 to 100. 0 μmol/L were added to the marginal cells suspension, the release of ATP was significantly decreased. However, with concentrations of aristolochic acid less than 100. 0 μmol/L, therelease of ATP tended to be not significantly different from the amount of ATP released by control group. Conclusions ATP could be release from marginal cells cultured in vitro of vascular stria in neonatal rats. ATP release from marginal cells has relevant with calcium pump, K ＋ channel state and signaling pathway related enzymes.