Chen I-Shu, Chou Chiang-Ting, Liang Wei-Zhe, Liu Yuan-Yuarn, Kuo Chun-Chi, Wang Jue-Long, Hao Lyh-Jyh, Jan Chung-Ren
Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung 81362, Taiwan, Republic of China.
Department of Nursing, Division of Basic Medical Sciences, Chang Gung University of Science and Technology, Chia-Yi 61363, Taiwan, Republic of China.
Chin J Physiol. 2018 Aug 31;61(4):221-229. doi: 10.4077/CJP.2018.BAH594.
Captopril, an angiotensin-converting enzyme (ACE) inhibitor, induced different Ca²⁺ signaling responses in various cell models. However, the effect of captopril on Ca²⁺ homeostasis and cell viability in hepatoma cells is unknown. This study examined whether captopril altered Ca²⁺ homeostasis and viability in HepG2 human hepatoma cells. Intracellular Ca²⁺ concentrations in suspended cells were monitored by using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1). Captopril at concentrations of 500-3000 μM induced [Ca²⁺]i rises in a concentration-dependent manner. Ca²⁺ removal reduced the signal by approximately 15%. Mn²⁺ has been shown to enter cells through similar mechanisms as Ca²⁺ but quenches fura-2 fluorescence at all excitation wavelengths. Captopril (3000 μM)-induced Mn²⁺ influx indirectly suggested that captopril evoked Ca²⁺ entry. Captopril-induced Ca²⁺ entry was inhibited by 15% by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and an inhibitor (GF109203X) and three inhibitors of store-operated Ca²⁺ channels: nifedipine, econazole and SKF96365. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished captopril-evoked [Ca²⁺]i rises. Conversely, treatment with captopril abolished BHQ-evoked [Ca²⁺]i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 70% of captopril-induced [Ca²⁺]i rises. Captopril at concentrations between 150-550 μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/AM (BAPTA/AM) did not reverse captopril’s cytotoxicity. Together, in HepG2 human hepatoma cells, captopril induced [Ca²⁺]i rises and caused cell death that was not triggered by preceding [Ca²⁺]i rises.
卡托普利是一种血管紧张素转换酶（ACE）抑制剂，在各种细胞模型中可诱导不同的Ca²⁺信号反应。然而，卡托普利对肝癌细胞中Ca²⁺稳态和细胞活力的影响尚不清楚。本研究检测了卡托普利是否会改变HepG2人肝癌细胞中的Ca²⁺稳态和活力。通过使用荧光Ca²⁺敏感染料fura-2监测悬浮细胞中的细胞内Ca²⁺浓度。使用4-[3-[4-碘苯基]-2-4(4-硝基苯基)-2H-5-四氮唑]-1,3-苯二磺酸盐]水溶性四氮唑-1（WST-1）检测细胞活力。浓度为500-3000μM的卡托普利以浓度依赖性方式诱导[Ca²⁺]i升高。Ca²⁺的去除使信号降低了约15%。已表明Mn²⁺通过与Ca²⁺相似的机制进入细胞，但在所有激发波长下都会淬灭fura-2荧光。卡托普利（3000μM）诱导的Mn²⁺内流间接表明卡托普利引起了Ca²⁺内流。蛋白激酶C（PKC）激活剂（佛波醇12-肉豆蔻酸酯13-乙酸酯，PMA）和抑制剂（GF109203X）以及三种储存-操作性Ca²⁺通道抑制剂：硝苯地平、酮康唑和SKF96365可使卡托普利诱导的Ca²⁺内流受到15%的抑制。在无Ca²⁺培养基中，用内质网Ca²⁺泵抑制剂2,5-二叔丁基对苯二酚（BHQ）处理可消除卡托普利引起的[Ca²⁺]i升高。相反，用卡托普利处理可消除BHQ引起的[Ca²⁺]i升高。用U73122抑制磷脂酶C（PLC）可抑制70%的卡托普利诱导的[Ca²⁺]i升高。浓度在150-550μM之间的卡托普利以浓度依赖性方式杀死细胞。用1,2-双（2-氨基苯氧基）乙烷-N,N,N’,N’-四乙酸/AM（BAPTA/AM）螯合胞质Ca²⁺并不能逆转卡托普利的细胞毒性。总之，在HepG2人肝癌细胞中，卡托普利诱导[Ca²⁺]i升高并导致细胞死亡，而这并非由先前的[Ca²⁺]i升高所触发。
