Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells
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Interplay between calcium ions (Ca2+) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However,details of the Ca2+ and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca2+ and ROS. Here, a real-time monitoring system for Ca2+and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca2+ 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 Ca2+ transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed,distinct Ca2+ and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca2+ and ROS.
Abstract
Interplay between calcium ions (Ca2+) and reactive oxygen species (ROS) delicately controls diverse pathophysiological functions of vascular smooth muscle cells (VSMCs). However,details of the Ca2+ and ROS signaling network have been hindered by the absence of a method for dual measurement of Ca2+ and ROS. Here, a real-time monitoring system for Ca2+and ROS was established using a genetically encoded hydrogen peroxide indicator, HyPer, and a ratiometric Ca2+ 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 Ca2+ transient and hydrogen peroxide were practically measurable in HyPer-expressing, fura-2-loaded VSMCs. Indeed,distinct Ca2+ and ROS signals could be successfully detected in serotonin-stimulated VSMCs. The system established in this study is applicable to studies of crosstalk between Ca2+ and ROS.
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