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KMID : 0624620130460120600
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BMB Reports
2013 Volume.46 No. 12 p.600 ~ p.605
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Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells
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Chang Kyung-Hwa
Park Jung-Min
Lee Moo-Yeol
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Abstract
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Interplay between calcium ions (Ca(2+)) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However, details of the Ca(2+) and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca(2+) and ROS. Here, a real-time monitoring system for Ca(2+) and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca(2+) indicator, fura-2. For the simultaneous detection of fura-2 and HyPer signals, 540 nm emission filter and 500 nm~ dichroic beamsplitter were combined with conventional exciters. The wide excitation spectrum of HyPer resulted in marginal cross-contamination with fura-2 signal. However, physiological Ca(2+) transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed, distinct Ca(2+) and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca(2+) and ROS.
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KEYWORD
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Calcium, Hydrogen peroxide, HyPer, Reactive oxygen species, Vascular smooth muscle cells
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