Abstract 5209: Oxidative Stress Uncouples eNOS in a BH4-independent Manner via the Formation of Protein Thiol-radicals
Imbalance of nitric oxide (NO) and superoxide (·O2−) production in the endothelium contributes to many cardiovascular diseases, including hypertension, atherosclerosis, and heart failure. Under conditions of oxidative stress, NO synthase (NOS) can switch from NO to ·O2− generation. We have demonstrated that endothelial NOS (eNOS) is reversibly S-glutathiolated in vivo and in vitro, in response to oxidative stress. This redox modification of eNOS leads to uncoupling, with decreased eNOS-derived NO and increased ·O2−. There are several proposed mechanisms that can lead to protein S-glutathiolation: thiol-disulfide exchange with GSSG; formation of thiyl radicals, sulphenic acid, or protein-S-nitrosothiols, which in turn react with GSH. Here we demonstrate the oxidant induced formation of an eNOS protein thiyl radical using immunoblotting, immunostaining, and mass spectrometry. BH4-free eNOS was used to self generate ·O2−, which in turn can modify sulfhydryl groups to form protein radicals. We trapped these short lived protein radicals with the spin trap DMPO. Immunoblotting using an anti-DMPO antibody, demonstrated the formation of eNOS protein radicals, which were abolished by SOD and L-NAME, and were Ca2+/CaM sensitive, indicating that protein radical formation is due to ·O2− generation from the eNOS heme domain. With BH4 reconstituted eNOS, which will only generate NO, formation of the eNOS protein radical is completely inhibited. Furthermore, in endothelial cells treated with menadione to trigger cellular ·O2− generation, formation of eNOS protein radical as visualized with confocal microscopy was increased compared to control, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Using mass spectrometric analysis, we identified Cys908 as the only residue involved in protein radical formation. Of note, we have also demonstrated the oxidant-induced S-glutathiolation of Cys908. Thus, eNOS protein radical formation provides the groundwork for a mechanism of ·O2−-directed regulation of eNOS, involving S-glutathiolation; defining a unique pathway for the redox regulation of cardiovascular function.
This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).