Mechanistic Insights into Inorganic Nitrite-Mediated Vasodilation of Isolated Aortic Rings under Oxidative/Hypertensive Conditions and S-Nitros(yl)ation of Proteins in Germ-Free Mice

Figure 1. Effects of different oxidants on endothelium-dependent vasodilation. (A) Pre-incubation with 1 mM hydrogen peroxide (H2O2) caused endothelial dysfunction (impaired acetylcholine [ACh] response) while 10 µM and 100 µM H2O2 showed no relevant effect. (B) Endothelium-dependent relaxation showed a concentration-dependent deterioration when incubating with the peroxynitrite donor Sin-1. (C) Treatment with 100 µM and 1 mM hypochlorous acid (HOCl) impaired ACh-response, while the lowest concentration of HOCl showed no difference to untreated rings. (D) Endothelium-dependent vasodilation was impaired by ATII-infusion for 7 days. Data are presented as mean ± SEM from n = 14–16 (A,B,D), 7–14 (C) aortic rings/group. p < 0.05 * vs. Ctr; # vs. 10 µM.

Figure 2. Effects of different oxidants on nitrite-dependent vasodilation. (A,B) All concentrations of hydrogen peroxide (H2O2) and the peroxynitrite donor Sin-1 had no effect on nitrite-induced vasodilation. (C) Nitrite-dependent aortic relaxation was not changed by low concentrations of hypochlorous acid (HOCl), while the highest concentration of HOCl (1 mM) clearly impaired vessel function. (D) Aortic rings of ATII-infused rats showed a worse response to nitrite than controls. Data are presented as mean ± SEM from n = 16 (A,B), 8–24 (C) and 15 (D) aortic rings/group. p < 0.05 * vs. Ctr; # vs. 10 µM; + vs. 100 µM.

Figure 3. Effects of Sin-1 incubation at different concentrations on XO/XDH expression. (A) Levels of xanthine dehydrogenase (XDH) showed a decreasing trend by Sin-1 treatment. (B,C) The conversion to aortic xanthine oxidase (XO) protein form showed an increasing trend at higher concentrations of Sin-1, resulting in a significantly higher XO/XDH ratio in all Sin-1 treated aortic rings compared to the control group. (D) Representative original Western blots used for the quantification of protein expression displayed in Figure 3A,B. Data are presented as mean ± SEM from n = 8–10 (A–D) aortic rings/group. p < 0.05 * vs. Ctr.

Figure 4. Effects of inorganic nitrite in vivo treatment on vasodilation and S-nitros(yl)ation of plasma proteins in the absence and presence of gut microbiota. (A,B) No changes in endothelium-dependent (ACh-response) and endothelium-independent (GTN-response) relaxation were observed after acute nitrite administration by gavage as compared with the solvent control (NaCl) in conventionally-raised (CONV-R) and germ-free (GF) mice. (C) Nitrite treatment resulted in increased plasma levels of S-nitrosothiol-positive proteins (by DMPO spin trapping); this effect was not present in GF animals. Data are presented as mean ± SEM from n = 6–14 (A,B) and 6–16 (C) animals/group. p < 0.05 * vs. NaCl; # vs. NO2−.