Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry
Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry
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DOI：
10.1007/s00210-007-0243-x
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发表时间：
2008-06-01
影响因子：
3.6
通讯作者：
Watanabe, Hiroshi
中科院分区：
文献类型：
--
作者：
Asai, Masayoshi;Takeuchi, Kazuhiko;Watanabe, Hiroshi
由于其高灵敏度、重复性和可靠性，Fura-2是测定钙离子水平的首选工具之一。最近，利用Fura-2/AM，一些钙通道阻滞剂(CCB)被报道对细胞内的钙信号有抑制作用，即使在不具有电压依赖性钙通道(VDCC)的非兴奋性细胞中也是如此。众所周知，一些CCB是光敏性的。由于光敏化学物质通常具有自发荧光性质，具有自发荧光的CCB很可能会干扰钙离子的荧光染料探针，这可能会导致对结果的误解。本研究的目的是阐明不同CCB在钙离子荧光探针上的特点，并为每个CCB确定合适的荧光探针，以便使用不具有VDCC的内皮细胞(ECs)进行可靠的钙测量。Thapsigargin(TG，内质网钙-ATPase抑制剂)使Fura-2 F340/F380比值从0.75+/-0.06增加到4.28+/-0.32，提示内皮细胞胞浆内钙离子浓度升高。维拉帕米、硝苯地平和尼卡地平均不影响TG诱导的内皮细胞Fura-2 F340/F380比值的升高，但氨氯地平剂量依赖性地抑制Fura-2比值在100 mM、1mM和10mM时分别增加24%、55%和82%。在无细胞体外实验中，由于氨氯地平在紫外线(UV光)激发下的自发荧光，氨氯地平本身对F380的增强作用远强于F340，从而导致Fura-2荧光的F340/F380比值降低。荧光探针Rhod-2/AM的激发波长不在氨氯地平的激发光谱范围内，结果表明氨氯地平对TG诱导的内皮细胞内钙离子升高无明显影响。当评估氨氯地平对细胞内钙离子浓度的影响时，由于氨氯地平和Fura-2之间存在荧光相互作用，因此不应使用Fura-2荧光分光光度法，强烈推荐使用Rhod-2。
Due to its high sensitivity, reproducibility and reliability, fura-2 is one of the most preferred tools to quantify Ca2+ levels. Recently, using fura-2/AM, some calcium channel blockers (CCBs) have been reported to have inhibitory effects on intracellular Ca2+ signaling even in nonexcitable cells that do not possess voltage-dependent Ca2+ channels (VDCC). Some CCBs are known to be photosensitive. Since photosensitive chemicals often have autofluorescent property, it is likely that CCBs with autofluorescence interfere with the fluorescent dye probes for Ca2+, which may cause misinterpretation of the results. The aim of this study was to clarify the characteristics of various CCBs on Ca2+ fluorescent probes and to identify proper fluorescent probes for each CCB to allow reliable Ca2+ measurement using endothelial cells (ECs), which possess no VDCC. Thapsigargin (TG, Ca2+-ATPase inhibitor on endoplasmic reticulum) increased fura-2 F340/F380 ratio from 0.75 +/- 0.06 to 4.28 +/- 0.32, suggesting the increase in cytosolic Ca2+ concentrations in ECs. Verapamil, nifedipine and nicardipine did not affect TG-induced increase in fura-2 F340/F380 ratio; however, amlodipine dose dependently inhibited the rise of fura-2 ratio by 24% at 100 nM, by 55% at 1 mu M and by 82% at 10 mu M, respectively, in ECs. In ex vivo experiments without cells, due to the autofluorescence of amlodipine excited by ultraviolet (UV light), amlodipine itself boosted the intensity of F380 much more than it did that of F340, which results in a decrease in F340/F380 ratio of fura-2 fluorescence. Rhod-2/AM, a fluorescent probe of which the excitation wavelength is out of the excitation spectrum of amlodipine, showed that amlodipine did not affect TG-induced intracellular Ca2+ increase in ECs. When the effect of amlodipine on intracellular Ca2+ concentration is assessed, fura-2 fluorospectrometry should not be used due to fluorescent interaction between amlodipine and fura-2, and using rhod-2 is strongly recommended.