Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite

Figures

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  Fig. 1. Sodium nitrate impairs the aortic vasorelaxing effects of sodium nitrite. Incubation of aortic rings with sodium nitrate 300 µmol/l shifted the concentration-response curve in response to sodium nitrite to the right, with significant decrease in pD2 (the negative logarithm of the concentration producing half maximum effect) and in the maximum response. Sodium nitrate 200 µM tended to exert similar effects. Panel a shows the relaxation response curves to cumulative concentrations of sodium nitrite under controls conditions (vehicle) and in the presence of sodium nitrate 200 and 300 µmol/l. Panel b shows pD2 values. Panel c shows the maximum effect. Data are shown as mean±S.E.M. (n=4–8/group).* P<0.05 versus the Control group.

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  Fig. 2. Sodium nitrate impairs the vasorelaxing effects of sodium nitrite in the mesenteric arterial bed. Nitrite decreased the perfusion pressure of mesenteric arterial bed contracted by phenylephrine in a concentration-dependent manner. Adding sodium nitrate 300 µmol/l to the perfusion solution attenuates this effect. The figure shows the % relaxation (% decrease in perfusion pressure) induced by increasing concentrations of sodium nitrite (from 0.1 µmol/l to 1 mmol/l) to the perfusion solution (Control), which is attenuated in the presence of sodium nitrate 300 µmol/l. Data are shown as mean±S.E. M. (n=7–8/group). *P<0.05 versus Control.

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  Fig. 3. Sodium nitrate attenuates the in vivo mean arterial blood pressure (MAP) responses to sodium nitrite in freely moving rats. The rats were made hypertensive by the acute administration of L-NAME (100 mg/kg, by gavage). Forty min later, the rats received nitrate (40 µmol/kg) or saline (controls) intravenously. Ten minutes later, increasing doses of sodium nitrite (1, 3, 10, 30 and 100 µmol/Kg) were infused intravenously every ten min. Panel a shows the MAP after each dose of nitrite in rats that received saline (open bars) or nitrate (closed bars). Panel b shows the changes in MAP after each dose of nitrite in rats that received saline (open bars) or nitrate (closed bars). Data are shown as mean±S.E.M. (n =5–6/group). P<0.001 for two-way ANOVA. *P<0.05 versus respective Control group.

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  Fig. 4. Plasma nitrite and nitrate concentrations in rats after intravenous administration. The rats received L-NAME (100 mg/kg, by gavage) followed 40 min later by sodium nitrate (40 µmol/kg) or saline intravenously, and then doses of sodium nitrite (1, 3, 10, 30 and 100 µmol/Kg) intravenously, every ten min. Panels a and b show nitrite and nitrate concentrations, respectively, at the end of the experiments. Plasma nitrite and nitrate were also measured in some untreated rats. Data are shown as mean±S.E. M. (n=5–6/group). *P<0.05 comparison between nitrate and saline treated rats.

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  Fig. 5. Sodium nitrate does not affect the vascular influx and accumulation of nitrite labeled with stable isotope (15N-nitrite). Aortic rings were incubated with 15N-nitrite 1, 10 and 100 µmol/l (or vehicle) and nitrate 300 µmol/l (or vehicle) for 30 min, and then washed three times with PBS. The vascular concentrations of 15N-2,3-naphthotriazole (15N-NAT) were measured by Ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS) after 15N-nitrite was converted to 15N-NAT. Data are shown as mean±S.E.M.(n=5–12/group). * P<0.05** P<0.01.

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  Fig. 6. Sodium nitrate does not affect the aortic vasorelaxing effects of the NO-donor diethylenetriamine NONOate (DETANONOate). Incubation of aortic rings with sodium nitrate 300 µmol/l did not affect the aortic relaxing responses to DETANONOate studied at 10−10–10−5 mol/l cumulative concentrations. Panel a shows virtually identical relaxation response curves to cumulative concentrations of DETANONOate under controls conditions (vehicle) and in the presence of sodium nitrate 300 µmol/l. Panel b shows pD2 values. Panel c shows the maximum effect. Data are shown as mean±S.E.M. (n=6/group).

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  Fig. 7. Nitrate inhibits XOR mediated nitrite reductase activity. Panel a shows the rate of XOR-mediated NO formation from 1 mmol/l sodium nitrite in the presence of nitrate 30 mmol/l, which is approximately 60% lower than that found with vehicle (Control). Panel b shows representative tracings of NO formation from nitrite. The black tracing (Control group) shows that XOR activates NO formation from nitrite. Adding nitrate 30 mmol/l approximately 20 min after the reaction started immediately inhibits NO production. The red tracing shows that the presence of nitrate 30 mmol/l at baseline conditions (Nitrate group) attenuates XOR-mediated NO formation from nitrite by approximately 60%. Adding the selective XOR inhibitor febuxostat (50 nmol/l) to the reaction further inhibited XOR-mediated NO production from nitrite. Data are shown as mean±S.E.M. (n=3). * P<0.01 versus Control.

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  Fig. 8. Michaelis-Menten saturation curve to show that sodium nitrate inhibits XOR-mediated nitrite reductase activity and NO production. The rate of NO formation versus nitrite concentrations are shown for Control (vehicle) and in the presence of sodium nitrate 15 mmol/l. Nitrate significantly shifted the Michaelis Menten saturation curve to the right. The maximum rate (Vmax) was not affected by nitrate, whereas the Michaelis constant (Km)increased by almost 3-fold (P<0.05). Data are shown as mean±S.E.M. (n=3–4).* P<0.01 for between curves comparison.