Sun He-Ying, Wang Ning-Ping, Halkos Micheal E, Kerendi Faraz, Kin Hajime, Wang Ruo-Xiang, Guyton Robert A, Zhao Zhi-Qing
Department of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA 30308-2225, USA.
Eur J Pharmacol. 2004 Feb 20;486(2):121-31. doi: 10.1016/j.ejphar.2003.12.016.
Although increased Na(+)/H(+) exchanger type-1 (NHE-1) activity has been implicated in the pathogenesis of myocardial infarction, the role of NHE-1 in induction of apoptosis, and the potential mechanisms involved have not been fully characterized. This study tested the hypothesis that NHE-1 activity is involved in hypoxia (H)/re-oxygenation (Re)-induced cardiomyocyte apoptosis by increasing mitochondrial Ca(2+) ([Ca(2+)]m). Primary cultured neonatal rat cardiomyocytes were subjected to 4.5 h of H followed by 12 h of Re. Relative to H alone, the level of X-rhod-1 acetoxymethyl (AM)-labeled [Ca(2+)]m was increased, and the frequency of cell death (propidium iodide (PI) staining) and apoptotic cells (terminal deoxynucleotidyl transferase (TdT)-mediated-UTP nick end labeling [TUNEL]), confirmed by Annexin-V, were augmented at the end of Re, along with appearance of cytosolic cytochrome c, activation of caspase-3, and increased ratio of Bax and Bcl-2. Addition of cariporide (20 micromol/l), a well-known NHE-1 inhibitor, to cultured cells before H significantly reduced [Ca(2+)]m, the number of PI and TUNEL positive cells relative to the levels at end of Re, but did not completely eliminate these changes compared to Sham control. There was a strong trend for attenuation in increased levels of [Ca(2+)]m, and the number of PI and TUNEL positive cells when same dose of cariporide was added only at Re, but the difference in these variables did not reach significance. In contrast, the levels of [Ca(2+)]m and the number of PI and TUNEL positive cells were significantly reduced to a level comparable to Sham control when cariporide (20 micromol/l) was administered before H and during Re, respectively, associated with a reduction in cytosolic cytochrome c, caspase-3 activity and ratio of Bax and Bcl-2. In conclusion, these data suggest that NHE-1 is involved in induction of cardiomyocyte apoptosis during both H and Re through a [Ca(2+)]m-dependent manner, thereby resulting in activation of cytochrome c-caspase-3 signaling pathways.
尽管1型钠氢交换体（NHE-1）活性增加与心肌梗死的发病机制有关，但NHE-1在诱导细胞凋亡中的作用以及潜在机制尚未完全阐明。本研究检验了以下假设：NHE-1活性通过增加线粒体钙（[Ca²⁺]m）参与缺氧（H）/复氧（Re）诱导的心肌细胞凋亡。原代培养的新生大鼠心肌细胞先经历4.5小时缺氧，随后再进行12小时复氧。与单纯缺氧组相比，X-罗丹明-1乙酰甲酯（AM）标记的[Ca²⁺]m水平升高，复氧结束时细胞死亡（碘化丙啶（PI）染色）和凋亡细胞（末端脱氧核苷酸转移酶（TdT）介导的dUTP缺口末端标记法[TUNEL]）的频率增加，膜联蛋白-V证实了这一点，同时伴有胞质细胞色素c的出现、半胱天冬酶-3的激活以及Bax与Bcl-2比值增加。在缺氧前向培养细胞中加入知名的NHE-1抑制剂卡立泊来德（20 μmol/L），可显著降低[Ca²⁺]m、PI和TUNEL阳性细胞数量，相对于复氧结束时的水平有所降低，但与假手术对照组相比，并未完全消除这些变化。仅在复氧时加入相同剂量的卡立泊来德，[Ca²⁺]m水平升高以及PI和TUNEL阳性细胞数量有减弱的强烈趋势，但这些变量的差异未达到显著水平。相比之下，分别在缺氧前和复氧期间给予卡立泊来德（20 μmol/L）时，[Ca²⁺]m水平以及PI和TUNEL阳性细胞数量显著降低至与假手术对照组相当的水平，同时伴有胞质细胞色素c、半胱天冬酶-3活性以及Bax与Bcl-2比值的降低。总之，这些数据表明，NHE-1通过依赖[Ca²⁺]m的方式参与缺氧和复氧过程中心肌细胞凋亡的诱导，从而导致细胞色素c-半胱天冬酶-3信号通路的激活。
