Amiloride和Ni^(2+)抑制缺血/复灌引起的大鼠心肌细胞内钙的增加(英文)

Inhibitors of Na^+/H^+ and Na^+/Ca^(2+) exchange depress intracellular calcium elevation induced by ischemia/reperfusion in rat cardiac myocytes

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 5 10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 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。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 。

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 +/Ca 2+ exchange to changes in intracellular calcium ([Ca 2+] i) during simulated ischemia and reperfusion in quiescent isolated rat cardiac myocytes. [Ca 2+] i was measured by laser confocal microscope using the fluorescent indicator Fluo 3 and expressed as the corrected intensity of Fluo 3 fluorescence. [Ca 2+] 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 [Ca 2+] i during simulated ischemia and reperfusion was suppressed by 100 μmol/L amiloride (inhibitor of Na +/H +exchanger), 5 mmol/L NiCl 2 (inhibitor of Na +/Ca 2+ exchanger) and calcium-free solution; [Ca 2+] 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 NiCl 2 during reperfusion. In addition, no calcium oscillation was observed in cells pretreated with amiloride. These results suggest that Na +/H +exchange and Na +/Ca 2+ exchange are activated during simulated ischemia in isolated quiescent cardiac myocytes, leading to the elevation of [Ca 2+] i induced by simulated ischemia and reperfusion.