title: Feasibility of simultaneous measurement of cytosolic calcium and hydrogen peroxide in vascular smooth muscle cells.
abstract: 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.
authors.forename: Kyung-Hwa
authors.initials: KH
authors.lastname: Chang
authors.forename: Jung-Min
authors.initials: JM
authors.lastname: Park
authors.affiliation: College of Pharmacy, Dongguk University, Goyang 410-820, Korea mlee@dongguk.edu.
authors.forename: Moo-Yeol
authors.initials: MY
authors.lastname: Lee
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002118:Calcium / Q000032:analysis*; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism*; D016257:Fura-2 / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000032:analysis*; D007477:Ions / Q000737:chemistry; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D007477:Ions; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D006861:Hydrogen Peroxide; D002118:Calcium; D016257:Fura-2