Misinterpretation of the effect of amlodipine on cytosolic calcium concentration with fura-2 fluorospectrometry

Abstract

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 μM and by 82% at 10 μM, 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.