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

Abstract

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 ( normalO2•-) production in fetal pulmonary artery endothelia cells (PAECs) has never been reported. The aim of this study is to assess mitochondrial normalO2•- production in cultured PAECs over time using a novel quantitative optical approach. The rate, the sources, and the dynamics of normalO2•- 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 normalO2•-. 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 fluorescence microscopy was used to monitor the dynamic changes in normalO2•- fluorescence intensity over time in PAECs. The transient behaviors of the fluorescence time course showed exponential increases in the rate of normalO2•- production in the presence of the ETC uncoupler or inhibitors. The most dramatic and the fastest increase in normalO2•- 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 normalO2•- production induced by PCP. The results showed that mitochondrial respiratory complexes I, III and IV are sources of normalO2•- production in PAECs, and a new observation that ROS production during uncoupling of mitochondrial respiration 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 vitro.

Time‐lapse microscopy of oxidative stress demonstrates metabolic sensitivity of retinal pericytes under high glucose condition

“…The slopes of the intensity right after the addition of mitochondrial metabolic modulators (t = 30 min) were compared statistically in Figure 3, demonstrating significant differences between REC and RPC. These slopes are correlated with the intracellular ROS initiation rate [6]. The red and blue bars in Figure 3 represent initial ROS production rate in REC and RPC, respectively.…”

“…The enhanced eNOS activity, and hence increased bioavailability of NO is consistent with the decreased generation rate of superoxide in REC under HG. Moreover, the increased production of eNOS is shown to prevent apoptosis of EC under HG . This may justify the decreased rate of apoptosis in REC compared to the RPC due to diabetes.…”

“…Lack of proton gradient for phosphorylation and polarized membrane potential activates a mechanism to compensate for the uncoupling effect of PCP, by increasing proton pumping and respiration, to reestablish the proton gradient. Ironically, the increase in the activity of the complexes I and II in the uncoupled chain , increases electron transfer along the electron transport chain and as a result, increases electron leak to oxygen leading to superoxide production . It is also worth noting that Cadenas et al analyzed the effect of protophores in relation to the propositions of a cyclic mechanism activated in complex II of the electron transport chain.…”

“…Our developed methodology to obtain final intensity profiles of the cells from the input fluorescent images was previously reported . The intensity profile of the red fluorescence images shows the dynamic of ROS production in response to MitoSOX ( t = 10 min) and metabolic stressors ( t = 30 min).…”

Overexpression of Myocardin-related transcription factor-A attenuated middle cerebral artery occlusion/reperfusion-induced apoptosis via the Mcl-1/Cyt C/cleaved caspase 3 pathway

“…The protein expression and colocalization were detected using a laser scanning confocal microscope (CX31-32RFL, Olympus). 29…”