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

Figure 6

PTH selectively impairs Bk signaling through oxidation of Bk receptor B2 (a–i). BAECs were treated with PTH (0.1 nM) or exposed to hypoxia, and mROS levels were determined incubating the cells with the fluorescent probe Mitosox. The fluorescence intensity was determined by citofluorimetry. The fluorescence relative to ROS levels was expressed as fold change vs. controls ( vs. CTRL, vs. CTRL). The image is the mean of three independent experiments (a). Representative images of flow cytometry of three independent experiments, evaluating mROS in response to PTH or hypoxia (b). The levels of protein oxidation in whole cell lysates of PTH pretreated or O₂-deprived BAECs were determined by OxyBlot Protein Detection Kit as described in Materials and Methods and showed as levels of 2,4-dinitrophenylhydrazine (DNP) bound to proteins (c). Densitometric analysis of protein oxidation in response to either PTH or hypoxia is reported as mean % of increase ± SD vs. CTRL ( vs. CTRL, vs. CTRL) (d). The B2 receptor and mAch receptors (M1-M3) were immunoprecipitated from cells treated with PTH for 24 h, and the levels of receptor oxidation were determined by OxyBlot Protein Detection Kit as described in Materials and Methods and shown as levels of 2,4-dinitrophenylhydrazine (DNP) bound to the B2 receptor (e), mAch M1 receptor (g), and mAch M3 receptor (h). CTRL− represents an immunoprecipitation using the secondary antibody to test signal specificity. CTRL+ represents a whole cell lysate. The images are representative of results from three independent experiments. Densitometric analyses of the oxidized B2 receptor (f) and mAch M1-M3 receptors (i) are shown in bar graphs as mean ± SD () of fold change vs. control. Both images are the mean of three independent experiments.