Abstract 5209: Oxidative Stress Uncouples eNOS in a BH4-independent Manner via the Formation of Protein Thiol-radicals

Abstract

Imbalance of nitric oxide (NO) and superoxide (·O 2 − ) 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 ·O 2 − 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 ·O 2 − . 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. BH 4 -free eNOS was used to self generate ·O 2 − , 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 Ca 2+ /CaM sensitive, indicating that protein radical formation is due to ·O 2 − generation from the eNOS heme domain. With BH 4 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 ·O 2 − 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 ·O 2 − -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).