Oxidative Medicine and Cellular Longevity / 2018 / Article / Fig 7

Research Article

Parathyroid Hormone Causes Endothelial Dysfunction by Inducing Mitochondrial ROS and Specific Oxidative Signal Transduction Modifications

Figure 7

The inhibition of mitochondrial Ca2+ uptake limits PTH-dependent mitochondrial ROS production and endothelial dysfunction (a–i). The functional interplay between Ca2+ signaling and ROS production is a well-established concept. Briefly, Ca2+ released from intracellular stores is partially uptaken through MCU by mitochondria where it stimulates the respiratory chain. As soon as mitochondria are overloaded with calcium, the higher rate of oxygen consumption results in increased respiratory chain electron leakage and mitochondrial ROS production. In turn, ROS further affects Ca2+ fluxes across membranes through the oxidation of key regulators of Ca2+ homeostasis, such as ryanodine receptors (RyR) and sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) (a). BAECs were acutely stimulated with PTH (0.1 nM), and both cytosolic (b) and mitochondrial (c) Ca2+ were evaluated with specific fluorescent probes, Fluo4-AM and Rhod-2 AM, respectively, through a microplate fluorescence reader. The fluorescence was corrected by the background signal derived from nonmarked cells. All data are reported as 0 ( = fluorescence signal of BAECs stimulated with PTH; 0 = fluorescence signal of unstimulated BAECs). In another set of experiments, BAECs were pretreated with Ru360 (10 μM), stimulated with PTH (0.1 nM), and as described above, mROS levels were determined. The data of fluorescence intensity were expressed as fold change vs. control whose mean value was set as 1 () (d). BAECs were pretreated with Ru360 (10 μM), then stimulated with PTH (0.1 nM) for 24 hours, and Ca2+ fluxes were assessed in response to Bk (30 nM), as described above. All data are reported as 0 ( = fluorescence signal of BAECs stimulated with Bk; 0 = fluorescence signal of unstimulated BAECs) (e). Peak of cytosolic Ca2+ increase was reported as fold changes vs. control, whose mean value was set as 1 () (f). Plateau amplitude of Ca2+ kinetics in response to Bk was evaluated as area under the curve (AUC) of the plateau phase (starting from 100 seconds after Bk administration). Data are reported as fold changes vs. control, whose mean value was set as 1 () (g). BAECs were pretreated with Ru360 (10 μM), then stimulated with PTH (0.1 nM) for 24 hours, and NO release was assessed in response to Bk (30 nM), as described above. All data are reported as 0 ( = fluorescence signal of BAECs stimulated with Bk; 0 = fluorescence signal of unstimulated BAECs) (h). Peak of NO release was reported as fold changes vs. control, whose mean value was set as 1 () (i). All images are representative of three independent experiments.
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