Lee Nai-Yuan, Sun Ming-Hui, Ko Wang-Sheng
Department of Medical Research, Kuang-Tien General Hospital, Taichung 43353, Taiwan, Republic of China.
Chin J Physiol. 2010 Oct 31;53(5):294-8. doi: 10.4077/cjp.2010.amk057.
A rise in cytosolic free Ca2+ is the immediate trigger for contraction in heart muscle. In the present study, we investigated changes of intracellular Ca2+ increased by potassium chloride (KCl) and phenylephrine (PE) under hyperglycemia in rat heart myoblast H9c2 cells (BCRC 60096), respectively. We employed the fluorescent Ca2+-indicator, fura-2, and digital imaging microscopy to measure [Ca2+]i in H9c2 cells. Cells were cultured in hyperglycemic (30 mM glucose) Dulbecco's Modified Eagle's Medium. The variation of [Ca2+]i induced by KCI and PE in hyperglycemia was examined, respectively. Moreover, tiron, one of the antioxidants, was pretreated in hyperglycemia-treated H9c2 cells to measure the role of free radicals in the changes of intracellular [Ca2+]i. An influx in intracellular Ca2+ induced by KCl or PE was observed in a dose-dependent manner and reached the highest concentration of 434 +/- 42.3 nM and 443 +/- 42.8 nM (n = 24 cells), respectively. Moreover, this increase of intracellular [Ca2+]i induced by KCl or PE was markedly reduced in cells exposed to hyperglycemia (434 +/- 42.3 vs. 1.26 +/- 0.21 nM and 443 +/- 42.8 vs. 2.54 +/- 0.25 nM, n = 24 cells, P < 0.001, respectively). Similar changes were not observed in cells received mannitol showing same osmolarity. However, the reduction of intracellular [Ca2+]i induced by hyperglycemia was abolished significantly in the presence of tiron. Our results suggest that an increase of intracellular Ca2+ by KCl or PE in heart cell was markedly reduced by hyperglycemic treatment; mediation of free radicals in this action can be considered because it was reversed in the presence of tiron.
胞质游离Ca2+浓度升高是心肌收缩的直接触发因素。在本研究中，我们分别研究了高血糖状态下大鼠心肌成肌细胞H9c2细胞（BCRC 60096）中氯化钾（KCl）和去氧肾上腺素（PE）所引起的细胞内Ca2+变化。我们使用荧光Ca2+指示剂fura-2和数字成像显微镜来测量H9c2细胞中的[Ca2+]i。细胞在高糖（30 mM葡萄糖）的杜氏改良 Eagle 培养基中培养。分别检测了高血糖状态下KCl和PE诱导的[Ca2+]i变化。此外，在高糖处理的H9c2细胞中预先加入抗氧化剂之一的钛铁试剂，以检测自由基在细胞内[Ca2+]i变化中的作用。观察到KCl或PE诱导的细胞内Ca2+内流呈剂量依赖性，分别达到最高浓度434±42.3 nM和443±42.8 nM（n = 24个细胞）。此外，在高糖环境中的细胞中，KCl或PE诱导的细胞内[Ca2+]i升高明显降低（434±42.3 vs. 1.26±0.21 nM以及443±42.8 vs. 2.54±0.25 nM，n = 24个细胞，P均<0.001）。在接受等渗甘露醇的细胞中未观察到类似变化。然而，在存在钛铁试剂的情况下，高血糖诱导的细胞内[Ca2+]i降低被显著消除。我们的结果表明，高糖处理显著降低了心脏细胞中KCl或PE所引起的细胞内Ca2+升高；考虑到在存在钛铁试剂时这种作用被逆转，自由基可能介导了这一过程。
