Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice

In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH₄) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1^-/-) and heterozygous mutant (hph-1 ^+/- mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH₄. In the cerebral microvessels of hph-1^+/- and hph-1^-/- mice, increased superoxide anion production was inhibited by supplementation of BH₄ or NOS inhibitor- L- N^G-nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH ₄, increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH₄ bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature. Uncoupled eNOS impairs NO signaling in cerebral microvasculature of hph-1 mice The effects of eNOS uncoupling on cerebral microvasculature has not been studied to date. We used the GTP cyclohydrolase-I-deficient hph-1 mice to demonstrate that reduced bioavailability of BH₄ causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.