Abstract
Exposure of endothelial cells (EC) and blood vessels to elevated glucose elicits the formation of reactive oxygen species, resulting in NO insufficiency and vascular dysfunction. Oxidative loss of 5,6,7,8-tetrahydrobiopterin (BH4), an essential endothelial NO synthase (eNOS) cofactor, has been implicated as a mechanism of diabetes-associated NO insufficiency. Experiments examined the possibility that intracellular accumulation of catalytically-incompetent 7,8,-dihydrobiopterin (BH2) effectively competes with BH4 for eNOS occupancy, driving uncoupled superoxide production in lieu of NO. BH2 was undetectable in murine EC cultured in low glucose (5 mM), but comprised 40% of total pterin (BH4 and more oxidized species) after 48 h exposure to diabetic glucose levels (30 mM) - high glucose did not attenuate total pterin. Concomitant with BH2 accumulation, A23187-evoked NO activity was dampened and superoxide production was accelerated - this was accompanied by evidence of peroxynitrite formation, via reaction of NO with superoxide. Superoxide overproduction was abolished with a selective NOS inhibitor and with eNOS gene silencing, implicating uncoupled eNOS as the source. BH4 oxidation in EC was prevented by supplementation with glutathione (GSH) and accelerated by GSH depletion, linking thiol reserves to intracellular pterin redox status. Progressive BH4 oxidation and protein tyrosine nitration were recapitulated in vivo in the Zucker Diabetic Fatty rat model of type II diabetes and the atherosclerotic ApoE knockout mouse. These findings implicate diminished intracellular BH4:BH2, rather than depletion of BH4 per se, as the trigger of endothelial cell dysfunction in chronic vascular conditions such as diabetes and atherosclerosis, where oxidative stress is a feature. Therapeutic approaches that can transiently re-couple eNOS would provide a means to break the cycle of endothelial dysfunction and lead to long-term reinstatement of vascular health. These data show that, Nω-hydroxy-L-arginine (NOHA) reacts with superoxide and other reactive oxygen species, releasing NO and/or NO-related species. Notably, while unable to synthesize NO from its normal substrate arginine, BH2-bound eNOS is still able to synthesize NO from the reaction intermediate NOHA. Administration of NOHA to our high glucose endothelial cell model is effective in restoring NO synthesis while simultaneously reducing O2-, thus restoring BH4 levels and normal endothelial function.