Characterization of cerebral microvasculature in transgenic mice with endothelium targeted over-expression of GTP-cyclohydrolase I

Abstract

Tetrahydrobiopterin (BH4) is a critical determinant of nitric oxide (NO) production by nitric oxide synthase (NOS) in the vascular endothelium and its biosynthesis is regulated by the enzymatic activity of GTP-cyclohydrolase I (GTPCH I). The present study was designed to determine the effects of endothelium-targeted overexpression of GTPCH I (eGCH-Tg) on murine cerebral vascular function. Endothelium targeted over-expression of GTPCH I was associated with a significant increase in levels of BH4, as well as its oxidized product, 7,8-dihydrobiopterin (7,8-BH2) in cerebral microvessels. Importantly, ratio of BH4 to 7,8-BH2, indicative of BH4 available for eNOS activation, was significantly increased in eGCH-Tg mice. However, expression of endothelial NOS, levels of nitrate/nitrite--indicative of NO production--remained unchanged between cerebral microvessels of wild-type and eGCH-Tg mice. Furthermore, increased BH4 biosynthesis neither affected production of superoxide anion nor expression of antioxidant proteins. Moreover, endothelium-specific GTPCH I overexpression did not alter intracellular levels of cGMP, reflective of NO signaling in cerebral microvessels. The obtained results suggest that, despite a significant increase in BH4 bioavailability, generation of endothelial NO in cerebral microvessels remained unchanged in eGCH-Tg mice. We conclude that under physiological conditions the levels of BH4 in the cerebral microvessels are optimal for activation of endothelial NOS and NO/cGMP signaling.

Keywords: Cerebral microvessel; Endothelial nitric oxide synthase; GTP-cyclohydrolase I; Tetrahydrobiopterin.

Figure 1

(A) Protein expression studies confirmed that GTPCH I was increased in cerebral microvessels of eGCH-Tg mice (*P<0.05; n=4 independent experiments). Endothelium-targeted over-expression of GTPCH I resulted in increased levels of tetrahydrobiopterin (BH₄) (B) and 7,8-dihydrobiopterin (7,8-BH₂) (C). The ratio of BH₄ to 7,8-BH₂ (D), indicative of net BH₄ bioavailable for eNOS activation, was also significantly increased in cerebral microvessels of eGCH-Tg mice (*P<0.05 vs. non-transgenic wild-type littermates (Non-Tg); n=5-7).

Figure 2

Representative Western blots and densitometric analysis demonstrating no difference in expression of NOS isoforms in cerebral microvessels of non-transgenic wild-type mice (Non-Tg) and transgenic mice with endothelium-targeted over-expression of GTPCH I (eGCH-Tg) (n=4 independent experiments, n.s.).

Figure 3

(A) Levels of total NOx (NO₂ + NO₃) and (B) cGMP, indicators of NO production, were not significantly different between cerebral microvessels of wild-type mice (Non-Tg) and transgenic mice with endothelium-targeted over-expression of GTPCH I (eGCH-Tg) (n=4-7, P>0.05).

Figure 4

Endothelium-targeted over-expression of GTPCH I (eGCH-Tg) did not affect either production of superoxide anions (A; n=7) or expression of antioxidant proteins (B; n=4 independent experiments) in mice cerebral microvessels (P>0.05).

Figure 5

Endothelium-dependent relaxations to acetylcholine in isolated basilar arteries of wild-type littermates (Non-Tg) and transgenic mice with endothelium-targeted over-expression of GTPCH I (eGCH-Tg) in the absence (A) or in the presence (B) of cell permeable superoxide dismutase mimetic Mn(III) tetra(4-benzoic acid) porphyrin chloride (MnTBAP; 10⁻⁵ mol/L). Please note that MnTBAP significantly improved impaired relaxations to acetylcholine in eGCH-Tg mice basilar arteries (P<0.05; ANOVA plus Bonferroni’s; n=4-5). The relaxations were expressed as percentage of the increase in intraluminal diameter from the diameter obtained after submaximal contraction to U46619.

Figure 6

(A) Endothelium-independent relaxations to NO donor DEA-NONOate and (B) concentration-dependent contractions to prostaglandin H₂/thromboxane A₂ analog U46619 in isolated basilar arteries of wild-type littermates (Non-Tg) and transgenic mice with endothelium-targeted over-expression of GTPCH I (eGCH-Tg). Please note that both of the vascular responses were not significantly different between both groups of mice (n=4-5, P>0.05; ANOVA plus Bonferroni’s). The contractions to U46619 or relaxations to DEA-NONOate were expressed as percentage of the decrease in the basal intraluminal diameter or of the increase in intraluminal diameter from the diameter obtained after submaximal contraction to U46619, respectively.