乌头碱对心肌细胞内ATP酶及相关离子的影响

Effects of aconitine on ATPase and related ions in cardiac myocytes

目的:研究乌头碱对心肌细胞内Ca2+-ATP酶、Ca2+-Mg2+-ATP酶和Na+-K+-ATP酶的活性;Na+、Ca2+、K+含量以及钙超载的影响;探讨乌头碱对乳鼠心肌细胞生物电活动的影响。方法:不同浓度的乌头碱处理原代心肌细胞,通过Na+、Ca2+、K+检测试剂盒和Ca2+-ATP酶、Ca2+-Mg2+-ATP酶和Na+-K+-ATP酶检测试剂盒检测以上各指标的改变;使用激光扫描共聚焦显微镜,检测在不同浓度乌头碱给药下钙瞬变的发生;并利用Real-time PCR法及Western blot技术对不同浓度乌头碱处理组进行mRNA及蛋白的检测。结果:乌头碱低(1μmol/L)、中(μmol/L)、高(10μmol/L)剂量组作用于心肌细胞后,细胞内Na+,Ca2+浓度上升,而K+浓度降低,并且不同程度的降低了Ca2+-ATP酶、Ca2+-Mg2+-ATP酶和Na+-K+-ATP酶活性;乌头碱低(1μmol/L)、中(5μmol/L)、高(10μmol/L)剂量组作用于心肌细胞后,各自与空白组相比,不同浓度乌头碱均可抑制钙瞬变浓度,高浓度乌头碱可以显著增加钙瞬变频率,以及增加钙离子浓度;乌头碱对SERCA2的mRNA与蛋白表达显著下调,对RyR2的mRNA与蛋白表达显著上调,结果具有浓度依赖性。结论:乌头碱在高剂量时对心肌细胞具有明显的毒性作用,毒性机制可能与调节心肌细胞膜以及细胞内各离子的浓度有关。乌头碱可以调节钙离子通道相关蛋白SERCA2、RyR2的mRNA与蛋白质水平的表达,这种调节作用可能与乌头碱的毒性机制有关。

Objective： To study effects of aconitine on activities of ATP enzymes,intracellular related ions and intracellular calcium transients in cardiomyocytes of neonatal rats. Methods： Treatment of primary myocardial cells with different concentrations of Aconitine. Changes of the indexes were detected by Na＋,Ca2 ＋,K＋detection kits and Ca2 ＋-ATP enzymes,Ca2 ＋-Mg2 ＋-ATP enzymes and Na＋-K＋-ATP enzyme detection kits. Laser scanning confocal microscopy was used to detect the occurrence of calcium transients under different concentrations of aconitine. Further with different concentrations of aconitine treated cardiac myocytes,RNA and total proteins were extracted to detect the regulating effect of aconitine on SERCA2、RyR2 mRNA and protein expressions with Real-time PCR and Western blot technology respectively.Results： Low（ 1μmol L-1）,middle（ 5μmol L-1） and high（ 10μmol L-1） doses of aconitine acted on cardiac myocyte,intracellular Na＋and Ca2 ＋concentrations increased,whereas K＋concentrations decreased,and Ca2 ＋,-ATP,Ca2 ＋-Mg2 ＋,-ATP and Na＋-K＋-ATP enzymes were reduced in varying degrees. Low（ 1 mol/L）,middle（ 5 mol/L） and high doses（ 10 mol/L） of aconitine groups were set in myocardial cells assay,compared with their respective control group,different concentrations of aconitine inhibited calcium transient concentration,high concentration of aconitine significantly increased the calcium transient frequency,and increased the concentration of calcium ion. Aconitine significantly down regulated expressions of mRNA and protein in SERCA2,and up-regulated expressions of mRNA and protein in RyR2. Results were concentration-dependent. Conclusion： High dose of aconitine has the obvious toxic effect on cardiac muscle cells,the mechanism of toxicity may be related to the regulation of myocardial cell membrane and the concentration of intracellular ions. Aconitine can regulate expressions of mRNA and protein of calcium channel associated protein SERCA2 RyR2,which may be related to the toxicity mechanism of aconitine.