Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells

• Published in: BMB reports Vol. 46; no. 12; pp. 600 - 605
• Main Authors: Chang, K.H., Dongguk University, Goyang, Republic of Korea, Park, J.M., Dongguk University, Goyang, Republic of Korea, Lee, M.Y., Dongguk University, Goyang, Republic of Korea
• Format: Journal Article
• Language: English
• Published: Korea (South) Korean Society for Biochemistry and Molecular Biology 01.12.2013; 생화학분자생물학회
• Subjects: Animals; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; CALCIO; CALCIUM; Calcium - analysis; Cells, Cultured; Cytosol - chemistry; Cytosol - metabolism; ESPECIES DE OXIGENO REACTIVO; Fura-2 - chemistry; HYDROGEN PEROXIDE; Hydrogen Peroxide - analysis; HyPer,Vascular smooth muscle cells; Ions - chemistry; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Male; Microscopy, Confocal; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; PEROXIDO DE HIDROGENO; PEROXYDE D'HYDROGENE; Rats; Rats, Sprague-Dawley; REACTIVE OXYGEN SPECIES; Signal Transduction; Spectrometry, Fluorescence; 화학
• ISSN: 1976-6696; 1976-670X
• DOI: 10.5483/BMBRep.2013.46.12.103

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. [