5,6,7,8-Tetrahydrobiopterin (BH₄) is an essential cofactor of nitric oxide synthases (NOSs). Oxidation of BH₄, in the setting of diabetes and other chronic vasoinflammatory conditions, can cause cofactor insufficiency and uncoupling of endothelial NOS (eNOS), manifest by a switch from nitric oxide (NO) to superoxide production. Here we tested the hypothesis that eNOS uncoupling is not simply a consequence of BH₄insufficiency, but rather results from a diminished ratio of BH₄vs. its catalytically incompetent oxidation product, 7,8-dihydrobiopterin (BH₂). In support of this hypothesis, [³H]BH₄binding studies revealed that BH₄and BH₂bind eNOS with equal affinity ( K_d≈ 80 nM) and BH₂can rapidly and efficiently replace BH₄in preformed eNOS-BH₄complexes. Whereas the total biopterin pool of murine endothelial cells (ECs) was unaffected by 48-h exposure to diabetic glucose levels (30 mM), BH₂levels increased from undetectable to 40% of total biopterin. This BH₂accumulation was associated with diminished calcium ionophore-evoked NO activity and accelerated superoxide production. Since superoxide production was suppressed by NOS inhibitor treatment, eNOS was implicated as a principal superoxide source. Importantly, BH₄supplementation of ECs (in low and high glucose-containing media) revealed that calcium ionophore-evoked NO bioactivity correlates with intracellular BH₄:BH₂and not absolute intracellular levels of BH₄. Reciprocally, superoxide production was found to negatively correlate with intracellular BH₄:BH₂. Hyperglycemia-associated BH₄oxidation and NO insufficiency was recapitulated in vivo, in the Zucker diabetic fatty rat model of type 2 diabetes. Together, these findings implicate diminished intracellular BH₄:BH₂, rather than BH₄depletion per se, as the molecular trigger for NO insufficiency in diabetes.

Asymmetric dimethylarginine (ADMA) is a naturally occurring inhibitor of nitric oxide synthesis that accumulates in a wide range of diseases associated with endothelial dysfunction and enhanced atherosclerosis. Clinical studies implicate plasma ADMA as a major novel cardiovascular risk factor, but the mechanisms by which low concentrations of ADMA produce adverse effects on the cardiovascular system are unclear. We treated human coronary artery endothelial cells with pathophysiological concentrations of ADMA and assessed the effects on gene expression using U133A GeneChips (Affymetrix). Changes in several genes, including bone morphogenetic protein 2 inducible kinase (BMP2K), SMA-related protein 5 (Smad5), bone morphogenetic protein receptor 1A, and protein arginine methyltransferase 3 (PRMT3; also known as HRMT1L3), were confirmed by Northern blotting, quantitative PCR, and in some instances Western blotting analysis to detect changes in protein expression. To determine whether these changes also occurred in vivo, tissue from gene deletion mice with raised ADMA levels was examined. More than 50 genes were significantly altered in endothelial cells after treatment with pathophysiological concentrations of ADMA (2 μM). We detected specific patterns of changes that identify pathways involved in processes relevant to cardiovascular risk and pulmonary hypertension. Changes in BMP2K and PRMT3 were confirmed at mRNA and protein levels, in vitro and in vivo. Pathophysiological concentrations of ADMA are sufficient to elicit significant changes in coronary artery endothelial cell gene expression. Changes in bone morphogenetic protein signalling, and in enzymes involved in arginine methylation, may be particularly relevant to understanding the pathophysiological significance of raised ADMA levels. This study identifies the mechanisms by which increased ADMA may contribute to common cardiovascular diseases and thereby indicates possible targets for therapies.