This study was supported by grants from National Natural Science Foundation, and the Science and Technology Committee of Shanghai Municipality (02JC14038).

Corresponding author. Tel: +86-21-54920305; Fax: +86-21-54920306; E-mail: znzhou@sever.shcnc.ac.cn

Research Paper

Inhibitors of Na+/H+ and Na+/Ca2+ exchange depress intracellular calcium elevation induced by ischemia/reperfusion in rat cardiac myocytes

DONG Jian-Wen, ZHU Hai-Feng, ZHOU Zhao-Nian*

Laboratory of Hypoxic Cardiovascular Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031

Abstract: An increase in cytosolic free calcium has been shown to occur during ischemia in perfused hearts and plays a pivotal role in ischemia/reperfusion injury. The objective of this study was to investigate the contributions of Na+/H+exchange and Na+/Ca2+ exchange to changes in intracellular calcium ([Ca2+]i) during simulated ischemia and reperfusion in quiescent isolated rat cardiac myocytes. [Ca2+]i was measured by laser confocal microscope using the fluorescent indicator Fluo 3 and expressed as the corrected intensity of Fluo 3 fluorescence. [Ca2+]i increased to 140.3±13.0% (P<0.05 vs preischemic control 100%) after 5 min simulated ischemia, and remained at high level of 142.8±15.5% (P<0.05) after the following 15 min reperfusion. The increase in [Ca2+]i during simulated ischemia and reperfusion was suppressed by 100 μmol/L amiloride (inhibitor of Na+/H+exchanger), 5 mmol/L NiCl2 (inhibitor of Na+/Ca2+ exchanger) and calcium-free solution; [Ca2+]i was 101.4±16.3%, 110.4±11.1% and 107.1±10.8%, respectively, after 5 min simulated ischemia, and 97.8±14.3%, 106.2±14.5% and 106.6±15.7%, respectively, after 15 min reperfusion. Compared with control cells, the amplitude of spontaneous calcium oscillation was lessened in cells treated with Ca-free perfusion and NiCl2 during reperfusion. In addition, no calcium oscillation was observed in cells pretreated with amiloride. These results suggest that Na+/H+exchange and Na+/Ca2+ exchange are activated during simulated ischemia in isolated quiescent cardiac myocytes, leading to the elevation of [Ca2+]i induced by simulated ischemia and reperfusion.

Key words: cardiac myocytes; ischemia/reperfusion; calcium; Na+/H+ exchanger; sodium-calcium exchanger

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

董建文, 朱海峰, 周兆年*

中国科学院上海生命科学研究院低氧心血管生理实验室, 上海 200031

摘要:本文旨在研究Na+/H+交换以及Na+/Ca2+交换对模拟缺血/复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血/复灌处理, 在缺血期间分别应用Na+/H+交换抑制剂阿米洛利（amiloride）、 Na+/Ca2+交换抑制剂NiCl2以及无钙液, 观察它们对细胞内游离Ca2+浓度变化的影响。利用Zeiss-LSM-510激光共聚焦显微镜检测、采集细胞内游离Ca2+的指示剂Fluo 3-AM的荧光信号, 计算出相对于正常(缺血前)的相对荧光强度, 以表示胞内游离Ca2+浓度的变化。结果显示, 模拟缺血引起大鼠心肌细胞内游离Ca2+持续上升, 缺血前的相对荧光强度值为100% , 模拟缺血5 min后为 140.3±13.0% (P<0.05), 复灌15 min后为 142.8±15.5% (P<0.05)。经100 μmol/L amiloride、 5 mmol/L NiCl2和无钙液分别预处理, 模拟缺血5 min后的相对荧光强度分别为101.4±16.3% (P<0.05)、 110.4±11.1% (P<0.05)和107.1±10.8 (P<0.05); 复灌15 min后则分别为 97.8±14.3% (P<0.05)、 106.2±14.5% (P<0.0 5)和106.6±15.7 (P<0.05)。另外, 与对照组细胞相比, 再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱, amiloride处理组细胞无明显钙振荡出现。上述结果表明, 在静息心肌细胞模拟缺血/再灌注损伤模型中, Na+/H+交换和Na+/Ca2+交换在缺血期被激活; Na+/H+交换和Na+/Ca2+交换的激活对于缺血复灌引起的细胞内钙的升高是非常重要的。

关键词: 心肌细胞; 缺血再灌注; 钙离子; Na+/H+交换; Na+/Ca2+交换

中图分类号: Q463