Redox regulation of the signaling pathways leading to eNOS phosphorylation

Oxidative stress rnediates positive and negative effects on physiological processes. Recent reports show that H2O2 induces phosphorylation and activation of endothelial nitric oxide synthase (eNOS) through an Akt-pliospliorylatioii-depeiideiit pathway. In this study, we assessed activation of eNOS and Akt by determining their phosphorylation status. Whereas moderate levels of H2O2 (100 mu M) activated the Akt/eNOS pathway higher levels (500 mu M) did not, suggesting differential effects by differing levels of oxidative stress. We then found that two pro-oxidants with activity on sulfydryl groups, 1-chloro-2,4-dinitrobenzene (CDNB) and diethyl maleate (DEM), blocked the phosphorlation events induced by 100 mu M H2O2. GSH was not a target thiol in this system because blithionine sulfoximine did not inhibit this phosphorylation. However, down-regulation of cell membrane Surface and intracellular free thiols was associated with the inhibition of phosphorylation, Suggesting that oxidation of non-GSH thiols inhibits the H2O2-induced phosphorylation of eNOS and Akt. DTT reversed the inhibitory effects of CDNB and DEM on Akt phosphorylation and concomitantly restored cell surface thiol levels more efficiently than it restored intracellular thiols. suggesting a more prominent rote for the former. Similarly, DEM and CDNB inhibited TNF-alpha-induced Akt and eNOS phosphorylation, suggesting that thiol modification is involved in eNOS inductive pathways. Our findings suggest that eNOS activation is exquisitely sensitive to regulation by redox and that cell surface thiols, other than glutathione, regulate signal transduction leading to phosphorylation of Akt and eNOS. Published bv Elsevier Inc.