Jan C R, Tseng C J, Chou K J, Chiang H T
Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan.
Life Sci. 2000;66(23):2289-96. doi: 10.1016/s0024-3205(00)00558-0.
The effect of clotrimazole on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ indicator. Clotrimazole (1-30 microM) induced a concentration-dependent [Ca2+]i increase. The [Ca2+]i increase comprised an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals by reducing both the initial rise and the decay phase, indicating that clotrimazole triggered both Ca2+ influx and Ca2+ release. Pretreatment with 30 microM clotrimazole in Ca2+-free medium abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and conversely, pretreatment with thapsigargin prevented clotrimazole from releasing more Ca2+. This suggests that the thapsigargin-sensitive Ca2+ store is the source of clotrimazole-induced Ca2+ release. Clotrimazole (10 microM) triggered Mn2+ quench of fura-2 fluorescence which was partly inhibited by 1 mM La3+. Addition of 3 mM Ca2+ induced a [Ca2+]i increase after preincubation with 10 microM clotrimazole in Ca2+-free medium, indicating that clotrimazole activated capacitative Ca2+ entry. However, 10 and 30 microM clotrimazole inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry by 21% and 74%, respectively. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 30 microM clotrimazole-induced Ca2+ release by 51%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This implies that clotrimazole induces Ca2+ release in an IP3-independent manner, which could be modulated by phospholipase A2-coupled events.
采用fura-2作为钙离子指示剂，研究了克霉唑对Madin Darby犬肾（MDCK）细胞钙离子信号的影响。克霉唑（1-30微摩尔）诱导细胞内钙离子浓度（[Ca2+]i）呈浓度依赖性升高。[Ca2+]i升高包括一个初始上升阶段和一个缓慢衰减阶段。去除细胞外钙离子通过减少初始上升阶段和衰减阶段部分抑制了钙离子信号，表明克霉唑触发了钙离子内流和钙离子释放。在无钙培养基中用30微摩尔克霉唑预处理消除了毒胡萝卜素（1微摩尔，一种内质网钙离子泵抑制剂）诱导的钙离子释放，相反，用毒胡萝卜素预处理可防止克霉唑释放更多钙离子。这表明毒胡萝卜素敏感的钙离子储存库是克霉唑诱导钙离子释放的来源。克霉唑（10微摩尔）触发了fura-2荧光的锰离子淬灭，1毫摩尔镧离子可部分抑制该淬灭。在无钙培养基中用10微摩尔克霉唑预孵育后，加入3毫摩尔钙离子诱导[Ca2+]i升高，表明克霉唑激活了容量性钙离子内流。然而，10和30微摩尔克霉唑分别抑制1微摩尔毒胡萝卜素诱导的容量性钙离子内流21%和74%。用40微摩尔马兜铃酸预处理以抑制磷脂酶A2可使30微摩尔克霉唑诱导的钙离子释放减少51%，但用2微摩尔U73122抑制磷脂酶C几乎没有效果。这意味着克霉唑以不依赖三磷酸肌醇（IP3）的方式诱导钙离子释放，这可能受磷脂酶A2偶联事件的调节。
