Jan C R, Yu C C, Huang J K
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Taiwan 813.
Anticancer Res. 2000 Nov-Dec;20(6B):4355-9.
The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride) (W-7), a widely used calmodulin inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in bladder female transitional cancer (BFTC) cells was examined using fura-2 as a Ca2+ dye. W-7 (10-1000 microM) caused an increase in [Ca2+]i in a concentration-dependent manner with an EC50 of 75 microM. The [Ca2+]i response was composed of an initial rise and a sustained plateau without significant decaying during the measurement of 250 seconds. Extracellular Ca2+ removal dramatically decreased the Ca2+ signals by 50-90%. W-7 (100 microM) failed to induce a [Ca2+]i increase in Ca(2+)-free medium after pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor; conversely, W-7 pretreatment abolished the Ca2+ release induced by thapsigargin. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 100 microM W-7 in Ca(2+)-free medium. W-7-induced Ca2+ release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17b-3-methoxyestra-1,3,5 (10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Together, this study shows that W-7 caused [Ca2+]i increases in human bladder cancer cells by releasing intracellular Ca2+ from the endoplasmic reticulum and also by causing extracellular Ca2+ influx with the later playing a dominant role. The W-7-induced intracellular Ca2+ release was uncoupled to a prior elevation in intracellular levels of inositol 1,4,5-trisphosphate (IP3).
使用fura-2作为钙离子指示剂，研究了广泛应用的钙调蛋白抑制剂N-(6-氨基己基)-5-氯-1-萘磺酰胺盐酸盐（W-7）对膀胱移行癌（BFTC）细胞内游离钙离子水平（[Ca2+]i）的影响。W-7（10 - 1000微摩尔）以浓度依赖的方式使[Ca2+]i升高，半数有效浓度（EC50）为75微摩尔。在250秒的测量过程中，[Ca2+]i反应由初始升高和持续平台期组成，无明显衰减。去除细胞外钙离子使钙离子信号显著降低50 - 90%。在用内质网钙离子泵抑制剂毒胡萝卜素（1微摩尔）预处理后，W-7（100微摩尔）未能在无钙培养基中诱导[Ca2+]i升高；相反，W-7预处理消除了毒胡萝卜素诱导的钙离子释放。在无钙培养基中用100微摩尔W-7预孵育后，添加3毫摩尔钙离子可使[Ca2+]i升高。用2微摩尔1-(6-((17β-3-甲氧基雌甾-1,3,5(10)-三烯-17-基)氨基)己基)-1H-吡咯-2,5-二酮（U73122）抑制磷脂酶C不会改变W-7诱导的钙离子释放。总之，本研究表明W-7通过从内质网释放细胞内钙离子以及引起细胞外钙离子内流使人类膀胱癌细胞中的[Ca2+]i升高，且后者起主要作用。W-7诱导的细胞内钙离子释放与细胞内肌醇1,4,5-三磷酸（IP3）水平的先前升高无关。
