Abstract
Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular homeostasis and generates nitric oxide (NO) by a process that is dependent on the binding of the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, electron transfer from NOS flavins becomes ‘uncoupled’ from l-arginine oxidation and superoxide is produced in place of NO. This process of eNOS ‘uncoupling’ is associated with many pathophysiologic conditions including atherosclerosis and hypercholesterolemia, although BH4 repletion only partially restores NOS activity and NOS-dependent vasodilation. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, however the exact mechanisms remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH; the rate-limiting enzyme in BH4 synthesis) and either WT or mutant eNOS rendered resistant to S-glutathionylation by mutation of two critical cysteine residues (C689S and C908S). We reveal that S-glutathionylation of eNOS by exposure of endothelial cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase (GR)-specific siRNA results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:BH2 ratio. In our model of BH4-dependent eNOS uncoupling, BCNU exposure further exacerbated superoxide production, BH4 oxidation and eNOS activity, effects that were totally abolished following mutation of either C689S or C908S. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling occur independently and their effects are additive.