PMC

Aortic BH₄ levels in Tg^SMCnox1 and WT mice treated with ANG II. BH₄ levels were quantified with the differential oxidation method and high-performance liquid chromatography. #P < 0.05 vs. BH₄ level in WT mice; *P < 0.05 vs. BH₄ level in untreated mice of the same genotype; +P < 0.01 vs. BH₄ level in WT ANG II; &P < 0.05 vs. oxidized BH₄ level in WT mice; xP < 0.05 vs. oxidized BH₄ level in Tg^SMCnox1 mice; ∼P < 0.05 vs. oxidized BH₄ level in WT ANG II mice.

Endothelial NO synthase (eNOS) protein expression. Representative Western blots (A) for dimer, monomer, and total eNOS using a low-temperature gel (3 different gels, but proteins from the same aortic homogenates). B: densitometry values for eNOS dimer/total eNOS ratio (n = 3). +P < 0.05 vs. untreated WT mice; *P < 0.001 vs. untreated WT mice; **P < 0.05 vs. WT ANG II; ∼P < 0.05 vs. WT ANG II; and #P < 0.01 vs. Tg^SMCnox1 mice with ANG II.

Effect of BH₄ treatment on superoxide production in Tg^SMCnox1 and WT mice infused with ANG II. Mice were infused with saline or ANG II (0.7 mg·kg⁻¹·day⁻¹) for 14 days. BH₄ was administered in pellets starting 2 days before ANG II. Intracellular superoxide level in aortic rings was measured by HPLC after incubation with hydroxyethidium. Values represent means ± SE for 5–10 animals/group. *P < 0.001 increase in superoxide level vs. saline-infused mice of the same genotype; #P < 0.01 vs. WT mice with the same treatment; +P < 0.01 decrease in superoxide vs. ANG II alone in the same genotype.

Effect of eNOS inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) on superoxide production in aortas from Tg^SMCnox1 and WT mice infused with ANG II. Mice were infused with saline or ANG II for 14 days. Aortic rings from the same mouse were incubated with or without l-NAME (0.1 mmol/l), and superoxide was measured by dihydroethidium-HPLC. Values represent means ± SE for 5–10 animals/group. +Significant change in superoxide in WT + l-NAME vs. WT (P < 0.05); *significant superoxide decrease in ANG II-infused WT + l-NAME vs. WT + ANG II (P < 0.05); #superoxide decrease in Tg^SMCnox1 infused with ANG II + l-NAME vs. Tg^SMCnox1 + ANG II (P < 0.005); ∼superoxide increase in Tg^SMCnox1 + ANG II vs. WT + ANG II not treated with l-NAME (P < 0.001).

Nitric oxide (NO) levels in Tg^SMCnox1 mice after ANG II-induced hypertension: effect of BH₄ supplementation. Calcium ionophore-stimulated NO production was estimated by ESR using the spin-trap iron diethyldithiocarbamate (Fe[DETC]₂). Four to five 2-mm segments of thoracic aorta were incubated with Fe[DETC]₂ and A-23187 (10 μmol/l) for 60 min, snap-frozen in liquid nitrogen, and subjected to ESR analysis. Mean data for 5–10 animals/group are shown. *Significant decrease in NO vs. saline-infused mice of the same genotype (P < 0.001); ∼significant increase in NO vs. saline-infused Tg^SMCnox1 (P < 0.01); #P < 0.05 vs. WT mice with the same treatment; +P < 0.05 vs. ANG II alone in mice of the same genotype.

Nox1 overexpression impairs endothelium-dependent vasorelaxation in a NADPH oxidase-dependent manner in mice. After the infusion of vehicle or ANG II for 13 days, vascular reactivity to the endothelium-dependent agonist acetylcholine (A-C) or endothelium-independent NO donor nitroglycerin (D) was measured in aortic rings from Tg^SMCnox1 and C57BL/6 mice. [ACh], acetylcholine concentration. Rings were preconstricted with PGF_2α (1 μmol/l). To study the effect of the NADPH oxidase inhibitor apocynin, rings from the same aortas were preincubated for 30 min with apocynin (0.05 mmol/l), which was added to the organ bath (B). In C, mice were treated with tetrahydrobiopterin (BH₄) in the food during ANG II or saline infusion before vasodilator measurements. Data are expressed as means ± SE. Lack of error bar indicates the error was smaller than the symbol; n = 4–10 mice/group. A: *P < 0.001, Tg^SMCnox1 with ANG II vs. WT and WT with ANG II; #P < 0.05, Tg^SMCnox1 with ANG II vs. Tg^SMCnox1; †P < 0.001, Tg^SMCnox1 vs. WT; NS indicates not significant. C: #P < 0.05, Tg^SMCnox1 with ANG II vs. Tg^SMCnox1; *P < 0.0001, Tg^SMCnox1 with ANG II vs. Tg^SMCnox1 with ANG II plus BH₄; †P < 0.0001, Tg^SMCnox1 vs. Tg^SMCnox1 treated with BH₄.

Production of aortic reactive oxygen species (ROS) in transgenic mice overexpressing NADPH oxidase (Nox) 1 in smooth muscle cells (Tg^SMCnox1) and wild-type (WT) mice. A: NADPH-dependent superoxide generation in aortas of Tg^SMCnox1 and C57BL/6 mice measured by electron spin oxidase microscopy (ESR) with 1-hydroxy-3-carboxypyrrolidine (CPH) as a spin probe. Mice were infused with saline or ANG II (0.7 mg·kg⁻¹·day⁻¹) for 14 days. Aortas were harvested, and membrane fractions were prepared by differential centrifugation. Superoxide production was measured using ESR after stimulation with 200 μmol/l of NADPH. Values were calculated as the difference of signals obtained from membranes in the presence and absence of NADPH. Values represent means ± SE for 6 animals/group. +Significant increase in superoxide level vs. saline-infused WT (P < 0.05); *significant increase in superoxide level vs. saline-infused WT mice (P < 0.001); #significant increase in superoxide level vs. saline-infused Tg^SMCnox1 mice (P < 0.001); ∼significant increase in superoxide level vs. ANG II-infused WT mice (P < 0.001). B: aortic H₂O₂ release in Tg^SMCnox1 and C57BL/6 mice. Amplex Red was used to measure the release of H₂O₂ in C57BL/6 mice (open bars) and Tg^SMCnox1 mice (filled bars) following infusion with saline or ANG II for 14 days. Values represent means ± SE for a minimum of 6 animals/group. *Significant increase in H₂O₂ level vs. saline-infused WT (P < 0.001); #significant increase vs. saline-infused Tg^SMCnox1 mice (P < 0.001); ∼significant increase vs. ANG II-infused WT mice (P < 0.001).