Chen Y C, Wang J L, Liu C P, Cheng J S, Chang H T, Yuk-Keung L, Su W, Law Y P, Chen W C, Jan C R
Department of Orthopaedic Surgery, Chang-Gung Memorial General Hospital-Kaohsiung, Taiwan, ROC.
Chin J Physiol. 2001 Jun 30;44(2):67-72.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca2+ indicator. Clomiphene at concentrations between 5-75 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 50 microM. The [Ca2+]i signal consisted of an initial rise and a sustained phase. Ca2+ removal reduced the Ca2+ signal by 40+/-10%. The [Ca2+]i increase induced by 50 microM clomiphene was inhibited by 80+/-5% by 10 microM nifedipine, but was insensitive to 50 microM La3+ or 10 microM verapamil. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; to uncouple mitochondria) inhibited 51+/-3% of 50 microM clomiphene-induced Ca2+ release; conversely, pretreatment with 50 microM clomiphene abolished the [Ca2+]i increase induced by thapsigargin, CCCP, and brefeldin A. The Ca2+ release-induced by 50 pM clomiphene was unchanged by inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Collectively, the results suggest that clomiphene increased [Ca2+]i, in osteoblast-like cells, by releasing intracellular Ca2+ in a phospholipase C-independent manner and by causing nifedipine-sensitive Ca2+ influx.
以fura - 2作为钙离子指示剂，研究了促排卵药物克罗米芬对MG63人骨肉瘤细胞胞质游离钙离子水平（[Ca2 +]i）的影响。浓度在5 - 75微摩尔之间的克罗米芬以浓度依赖的方式增加[Ca2 +]i，半数有效浓度（EC50）为50微摩尔。[Ca2 +]i信号包括一个初始上升阶段和一个持续阶段。去除钙离子可使钙离子信号降低40±10%。10微摩尔硝苯地平可使50微摩尔克罗米芬诱导的[Ca2 +]i增加受到80±5%的抑制，但对50微摩尔La3 +或10微摩尔维拉帕米不敏感。在无钙培养基中，用50微摩尔布雷菲德菌素A（破坏高尔基体钙离子储存）、1微摩尔毒胡萝卜素（抑制内质网钙离子泵）和羰基氰化物间氯苯腙（CCCP；使线粒体解偶联）预处理可抑制50微摩尔克罗米芬诱导的钙离子释放的51±3%；相反，用50微摩尔克罗米芬预处理可消除毒胡萝卜素、CCCP和布雷菲德菌素A诱导的[Ca2 +]i增加。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后，50皮摩尔克罗米芬诱导的钙离子释放不变。总的来说，结果表明克罗米芬通过以磷脂酶C非依赖的方式释放细胞内钙离子并引起硝苯地平敏感的钙离子内流，从而增加成骨样细胞中的[Ca2 +]i。
