Figure 2.
Ang II induces eNOS‐derived O2•− generation through glutathionylation. A, Effect of Ang II on O2•−‐sensitive DHE fluorescence. Cell nuclei were counterstained with DAPI (blue). Preincubation with l‐NAME (1 mmol/L) or DTT (1 mmol/L) is as indicated (n=9 control, 9 Ang II, and 6 in other groups; cells from 4 randomly chosen regions of interest in each group were included in the analysis). B, HPLC analysis of DHE oxidation products to separate the specific product (2‐OH‐E+) from the nonspecific product (E+) of DHE oxidation. [2‐OH‐E+] is calculated as the mean of values determined by fluorescence and electrochemical detectors, normalized to total protein concentration (pmol·mg−1·mL−1). C, Ang II‐induced O2•− as detected by HPLC analysis of specific DHE oxidation product (2‐OH‐E+) in HUVECs transfected with WT eNOS (WT) and double Cys mutant eNOS (double Cys mutant). Statistical comparison was made in each cell type between conditions of with and without Ang II (n=3). AU=arbitrary unit. Results are shown as means±SEM. Statistical significance, compared to control (P<0.05), is indicated by asterisk (*). In Figure 2A and 2B, statistical significance, compared to Ang II (P<0.05), is indicated by pound sign (#).DHE indicates dihydroethidium; DTT, dithiotreitol; eNOS, endothelial nitric oxide synthase; GSH, glutathione; HPLC, high performance liquid chromatography; HUVECs, human umbilical vein endothelial cells; PE, phenylephrine; WT, wild type.