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

Abstract

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 (BH(4)) 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 BH(4). In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) 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 BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.

Figure 1

Protein expression studies demonstrating expression of NO synthase in the microvessels and brain. Expression of neuronal markers, nNOS and NeuN were not detected in purified microvessels, while nNOS and NeuN could be detected in the brain. Protein expression of iNOS was significantly lower in purified microvessels as compared to brain. Protein expression studies were performed by SDS-PAGE on 25 μg total protein in brain and microvessels, and data are representative of expression from three different wild-type (C57BL/6) mice.

Figure 2

Enzymatic activity of GTP cyclohydrolase I (A) and levels of tetrahydrobiopterin (BH₄, B) were significantly decreased in the cerebral microvessels of hph-1^-/- and hph-1^+/- mice as compared to their sex-matched controls (* P<0.05, n=6 in comparison to wild-type male mice; ^#P<0.05, n=8 in comparison to wild-type female mice). Levels of 7,8-dihydrobiopterin (BH₂), oxidized product of BH₄ did not significantly increase in the cerebral microvessels of either hph-1^-/- or hph-1^+/- mice (C). In the cerebral microvessels of both male and female mice, ratio of BH₄ to BH₂, indicator of BH₄ bioavailable for eNOS activation, were significantly decreased in the cerebral microvessels of hph-1^-/- and hph-1^+/- mice (* P<0.05, n=6 in comparison to wild-type male mice; ^#P<0.05, n=8 in comparison to wild-type female mice).

Figure 3

Representative Western blots and densitometric analysis demonstrating expression of eNOS in the cerebral microvessels of male and female, hph-1^-/- and hph-1^+/- mice. Expression of eNOS did not differ between these three experimental groups (n=6).

Figure 4

(A) Superoxide anion production in the cerebral microvessels of hph-1^-/- and hph-1^+/- mice. Basal superoxide production between wild-type male and female mice was not statistically significant. Superoxide anion production remained significantly increased in male and female cerebral microvessels in both hph-1^-/- and hph-1^+/- groups (* P<0.05, in comparison to their wild-type controls, n=8). Treatment with L-NAME (30 μmol/l) significantly reduced the superoxide anion production in cerebral microvessels of both hph-1^-/- and hph-1^+/- mice (^# P<0.05 in L-NAME treated group as compared to corresponding sex-matched vehicle (Krebs-HEPES buffer) treatment, n=8). (B) Superoxide anion production in microvessels of female wild-type, hph-1^-/- and hph-1^+/-mice injected with BH₄ (100 μmol/kg, 3hours). Treatment with BH₄ abolished the increase in superoxide anion production observed in cerebral microvessels of hph-1^-/-and hph-1^+/- mice. Treatment with L-NAME (30 μmol/l) did not have any further effect on the superoxide anion production (P=ns, n=4).

Figure 4

(A) Superoxide anion production in the cerebral microvessels of hph-1^-/- and hph-1^+/- mice. Basal superoxide production between wild-type male and female mice was not statistically significant. Superoxide anion production remained significantly increased in male and female cerebral microvessels in both hph-1^-/- and hph-1^+/- groups (* P<0.05, in comparison to their wild-type controls, n=8). Treatment with L-NAME (30 μmol/l) significantly reduced the superoxide anion production in cerebral microvessels of both hph-1^-/- and hph-1^+/- mice (^# P<0.05 in L-NAME treated group as compared to corresponding sex-matched vehicle (Krebs-HEPES buffer) treatment, n=8). (B) Superoxide anion production in microvessels of female wild-type, hph-1^-/- and hph-1^+/-mice injected with BH₄ (100 μmol/kg, 3hours). Treatment with BH₄ abolished the increase in superoxide anion production observed in cerebral microvessels of hph-1^-/-and hph-1^+/- mice. Treatment with L-NAME (30 μmol/l) did not have any further effect on the superoxide anion production (P=ns, n=4).

Figure 5

Representative Western blots and densitometric analysis demonstrating expression of antioxidant proteins in the cerebral microvessels of both male and female, hph-1^-/- and hph-1^+/- mice. Expressions of CuZn superoxide dismutase (CuZnSOD), MnSOD and catalase did not differ in cerebral microvessels of hph-1^-/- and hph-1^+/- mice, as compared to their sex-matched wild-type controls (n=6).

Figure 6

Expression of 3-nitrotyrosine in the cerebral microvessels of both male and female, hph-1^-/- and hph-1^+/- mice. Densitometric analysis showed that 3-nitrotyrosine expression was significantly increased in both hph1^-/- and hph1^+/- mice (*P<0.05, n=4).

Figure 7

Levels of nitrite/nitrate, indicator of NO production, was significantly reduced in cerebral microvessels of hph-1^-/- and hph-1^+/- mice as compared to their sex-matched wild-type control mice (* P<0.05, n=6 in comparison to wild-type male mice; ^#P<0.05, n=6 in comparison to wild-type female mice).

Figure 8

Bar diagram depicting significantly reduced levels of cGMP, second messenger of NO, in cerebral microvessels of hph-1^-/- and hph-1^+/- mice as compared to their sex-matched wild-type control mice (* P<0.05, n=6 in comparison to wild-type male mice; ^#P<0.05, n=6 in comparison to wild-type female mice).