Abstract 5208: S-glutathiolation Uncouples Endothelial Nitric Oxide Synthase: A Novel Mechanism of Redox Signaling and Vascular Regulation

Abstract

Superoxide (·O 2 − ) and secondary oxidants are critical mediators of cellular signaling, proliferation and apoptosis. In response to oxidative stress, protein thiols undergo S-glutathiolation forming mixed disulfides and this modification plays an important role as an adaptive response to oxidative injury in cells, controlling cellular signaling in a manner similar to protein phosphorylation. We investigated the effects of S-glutathiolation on the regulation of eNOS function in vitro . We observe that S-glutathiolation greatly reduces NO synthase activity (70% ± 1%), and this loss of activity was reversed by DTT. In contrast, only a 56%±0.9% decrease in NADPH consumption was seen that was only partially inhibited by L-NAME or the calcium chelator EGTA, implicating an increase in eNOS derived ·O 2 − . Using EPR spin-trapping to directly measure eNOS derived ·O 2 − , we proved that S-glutathiolation greatly increased ·O 2 − generation from the enzyme. S-glutathiolation of human eNOS increased ·O 2 − generation >5-fold and L-NAME and EGTA only partially blocked this ·O 2 − generation. Thus, S-glutathiolation uncouples eNOS greatly increasing ·O 2 − generation and the partial or complete lack of inhibition of this by L-NAME and EGTA indicates that the observed ·O 2 − is largely derived from the reductase domain. Mass spectrometric analysis and molecular modeling revealed that two sights of S-glutathiolation in eNOS are two highly conserved cysteines found at the interface between the FMN and FAD-binding domains, C 689 and C 908 . The modification of these two cysteins appears to interrupt electron transfer to the oxygenase domain, leading to electron leakage. Furthermore, we observe that BCNU and H 2 O 2 - derived oxidative stress induces S-glutathiolation of eNOS in endothelial cells, with measured loss of NO and gain of ·O 2 − generation, and we show that this process impairs endothelium-dependent vasodilation. Additionally, we observe that in hypertensive vessels eNOS-S-glutathiolation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents. Thus, S-glutathiolation of eNOS serves as a pivotal switch that provides redox regulation of endothelial function and vascular tone. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).