Quantitative optical measurement of mitochondrial superoxide dynamics in pulmonary artery endothelial cells  

作　　者：Zahra Ghanian Girija Ganesh Konduri Said Halim Audi Amadou K.S.Camara Mahsa Ranji 

机构地区：[1]Department of Electrical Engineering University of Wisconsin-Milwaukee,Milwaukee,Wisconsin,USA [2]Department of Pediatrics,Division of Neonatology Medical College of Wisconsin,Milwaukee,Wisconsin,USA [3]Department of Biomedical Engineering,Marquette University Milwaukee,Wisconsin,USA [4]Department of Anesthesiology and Anesthesia Research Medical College of Wisconsin,Milwaukee,Wisconsin,USA

出　　处：《Journal of Innovative Optical Health Sciences》2018年第1期90-105,共16页创新光学健康科学杂志（英文）

基　　金：supported by a grant from UWM research growth initiative(101×290)to MR,grants R01 HL057268 and Muma Endowed Chair in Neonatology to GGK,NIH grant P01-GM-066730-12 to AKSC,and NIH grant 1R15HL129209 to SHA.

摘　　要：Reactive oxygen species(ROS)play a vital role in cell signaling and redox regulation,but when present in excess,lead to numerous pathologies.Detailed quantitative characterization of mitochondrial superoxide anion(O^(·-)_(2))production in fetal pulmonary artery endothelia cells(PAECs)has never been reported.The aim of this study is to assess mitochondrial O^(·-)_(2)pro-duction in cultured PAECs over time using a novel quantitative optical approach.The rate,the sources,and the dynamics of O^(·-)_(2)production were assessed using targeted metabolic modulators of the mitochondrial electron transport chain(ETC)complexes,specifically an uncoupler and inhibitors of the various ETC complexes,and inhibitors of extra-mitochondrial sources of O^(·-)_(2).After stabilization,the cells were loaded with nanomolar mitochondrial-targeted hydroethidine(Mito-HE,MitoSOX)online during the experiment without washout of the residual dye.Time-lapse fuorescence microscopy was used to monitor the dynamic changes in O^(·-)_(2)fluorescence intensity over time in PAECs.The transient behaviors of the fuorescence time course showed exponential increases in the rate of O^(·-)_(2) production in the presence of the ETC uncoupler or inhibitors.The most dramatic and the fastest increase in O^(·-)_(2)production was observed when the cells were treated with the uncoupling agent,PCP.We also showed that only the complex IV inhibitor,KCN,attenuated the marked surge in O^(·-)_(2)production induced by PCP.The results showed that mitochondrial respiratory complexes I,III and IV are sources of O^(·-)_(2) production in PAECs,and a new observation that ROS production during uncoupling of mitochondrial res-piration is mediated in part via complex IV.This novel method can be applied in other studies that examine ROS production under stress condition and during ROS mediated injuries in vritro.

关 键 词：Fluorescence microscopy time-lapse imaging oxidative stress superoxide pentachlorophenol sodium salt ROTENONE antimycin A potassium cyanide MitoSOX. 

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