Phosphorylation of endothelial nitric oxide synthase is increased in mouse model of GTP‐cyclohydrolase I deficiency

Abstract

Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial nitric oxide synthase (eNOS). Suboptimal level of BH4 reduces formation of nitric oxide leading to uncoupling of eNOS. However, the exact molecular mechanisms underlying the effects of eNOS uncoupling on vascular endothelium in‐vivo are not completely understood. In the present study, we used the hph‐1 mouse, which displays GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that BH4 levels were significantly reduced in the aortas of hph‐1 mice (3.9±0.5 pmol/mg; P<0.05 vs. wild‐type mice: 5.9±0.6 pmol/mg), while oxidative products of BH4, 7,8‐dihydrobiopterin (7,8‐BH2), were unchanged. Consequently, the BH4 to 7,8‐BH2 ratio was significantly reduced in the aorta of hph‐1 mice (P<0.05). Expression of eNOS protein was not different between wild‐type and hph‐1 mice. Unexpectedly, protein expressions of phosphorylated Ser1177‐eNOS and Ser473‐Akt1 were significantly increased in hph‐1 mice aortas (P<0.05). Consistent with uncoupling of eNOS, L‐NAME sensitive superoxide anion production and 3‐nitrotyrosine formation were significantly increased in aortas of hph‐1 mice (P<0.05). Protein expressions of CuZnSOD, MnSOD, and EC‐SOD were unaltered in the aortas of hph‐1 mice as compared with wild‐type mice. Our results suggest that uncoupling eNOS increases phosphorylation of Akt1 and eNOS. This effect is most likely mediated by increased formation of eNOS‐derived reactive oxygen species including superoxide anion and hydrogen peroxide.