Decreased Nitric Oxide Bioavailability in Diabetic Retinopathy: Involvement of Arginase Activity

Abstract

Purpose: : Significant impairment of endothelial cell (EC)-dependent vasorelaxation has been demonstrated in retinal arteries of STZ-induced diabetic rats as compared with non-diabetic controls, suggesting a decrease in availability of the vasodilator molecule nitric oxide (NO). However, numerous studies have shown that levels of NO products (nitrate/nitrite and the peroxynitrite biomarker nitrotyrosine) are increased in models of diabetic retinopathy (DR). This study was undertaken to assess levels of bioavailable NO during DR and to determine the role of the arginase enzyme, which competes with NO synthase for L-arginine, in uncoupling NOS and reducing bioavailable NO during DR.

Methods: : Experiments were performed in streptozotocin-induced diabetic rats and mice and in bovine retinal ECs treated with normal glucose (NG) or high glucose (25 mM, HG).

Results: : Measurement of nitrotyrosine and nitrate/nitrite levels confirmed significant increases in total NO products in the diabetic retinas. However, levels of the NO biomarkers nitrite and S-nitrosylated cysteine were reduced by diabetes. Imaging analysis using the NO indicator DAF-2DA and the superoxide detector DHE showed significant decreases in NO formation in the diabetic retinas that were accompanied by increases in superoxide formation. Immunolocalization studies showed prominent increases in arginase I in adjacent sections from the diabetic retinas. Parallel studies in knockout mice showed that arginase gene deletion enhanced NO formation and reduced superoxide formation in the diabetic retinas. In cultured ECs, HG treatment increased arginase activity and reduced nitrite release. Treatment with the specific arginase inhibitor [S]-[2-boronoethyl]-L-Cysteine-HCl restored the NO production.

Conclusions: : Diabetes-induced increases in oxidative/nitrosative stress are associated with increases in arginase I expression, decreases in bioavailable NO and increases in superoxide formation. Our results suggest a role for arginase in DR by uncoupling NOS and negatively regulating NO production.

Keywords: nitric oxide • diabetic retinopathy • oxidation/oxidative or free radical damage