细胞外低渗诱导大鼠胚胎心肌细胞H9c2容积激活性氯电流和调节性细胞容积回缩

Hypotonic challenges induce volume-activated chloride currents and regulatory volume decrease in rat embryonic myocardial H9c2 cells

目的:研究细胞外低渗诱导的大鼠胚胎心肌细胞(H9c2)容积激活性氯电流和调节性容积回缩(regulatory volume decrease,RVD)。方法:采用全细胞膜片钳技术记录低渗激活的H9c2细胞氯电流并分析电流特性;用实时活细胞影像系统拍摄细胞图像,测量细胞容积,探讨氯通道在H9c2细胞调节性容积回缩(RVD)过程中的作用。结果:等渗灌流下,可在H9c2细胞记录到一个较小的背景电流。47%低渗液灌流可迅速诱发一个具有外向优势的电流,该电流无明显时间依赖性失活和电压依赖性失活;在+80 mV和-80 mV电压钳制下,细胞的平均电流密度分别为(47.77±3.80)pA/pF和(-33.36±2.80)pA/pF;翻转电位为(-9.02±0.61)mV,接近氯离子的平衡电位(-0.9 mV)。高渗灌流液可以完全抑制该电流。此外,该电流可被氯通道阻断剂他莫昔芬、5-硝基-2-(3-苯丙胺)苯甲酸(NPPB)和ATP不同程度抑制。同时,细胞外灌流47%低渗液可诱发H9c2细胞产生RVD,100μmol/L的NPPB几乎完全抑制低渗诱发的RVD。结论:细胞外低渗刺激可以诱导H9c2细胞容积激活性氯电流和RVD。容积激活性氯通道在H9c2细胞RVD中起重要作用。

To investigate the volume - activated chloride currents and regulatory volume decrease （RVD） induced by hypotonic challenges in rat embryonic myocardial cell line H9c2. METHODS： The technique of whole -cell patch -clamp was used to record the chloride currents induced by hypotonic challenges and to clarify the properties of the currents in H9c2 cells. The changes of cell volume were observed by the technique of real - time living cell imaging, and the roles of chloride channels in RVD were analyzed. RESULTS： A weak background current was recorded in H9c2 cells under isotonic condition. Extracellular application of 47% hypotonic solution rapidly activated an outward rectified current, which did not exhibit time - and voltage - dependent inactivation with the current density of （47.77 ± 3.80） pA/pF at ＋ 80 mV and （ - 33.36 ~ 2.80） pA/pF at - 80 mV. The reversal potential was （ - 9.02 ± 0.61 ） mV, closed to the calculated equilibrium potential for C1- （ -0.9 mV）. The current was volume - sensitive and was completely sup- pressed by 47% hypertonic solution. In addition, chloride channel blockers tamoxifen （20 μmol/L）, 5 -nitro -2 - （3 - phenylpropylamino） benzoic acid （NPPB, 100 p^mol/L） and ATP （10 mmol/L） significantly inhibited the current with dif- ferent inhibitory ratios. The phenomenon of RVD was also observed in H9c2 cells under the condition of perfusion with 47% hypotonic solution. The chloride channel blocker NPPB at concentration of 100 μmol/L completely inhibited the RVD process. CONCLUSION： The volume -activated chloride channels, which are activated by extracellular hvootonic chal-lenges, play an important role in the process of regulatory volume decrease in H9c2 cells.