Oral nitrite treatment increases S-nitrosylation of vascular protein kinase C and attenuates the responses to angiotensin II

Fig. 1. Pretreatment with nitrite attenuates the blood pressure responses to angiotensin II in vivo and decreases angiontensin II-induced vascular contraction ex vivo. Panel A shows the increases in mean arterial blood pressure (MAP) after infusion of angiotensin II (0.03, 0.1, 0.3, 1.0 and 3.0 μg/kg, i.v.) in rats pretreated with oral nitrite (15 mg/kg) or vehicle for five days. Panel B shows the concentration-effect curves in response to angiotensin II (from 10−10 to 10−7 mol/l) using aortas from rats pretreated with oral nitrite (15 mg/kg) or vehicle for five days. Panels C and D show the maximum effect and the pD2 values (the negative logarithm of the concentration producing half maximum effect), respectively. Data are shown as mean ± S.E.M. (n = 8–10/group for Panel A, and n = 6/group for Panels B,C and D). *P < 0.05 versus Vehicle. #P < 0.05 versus Vehicle for the concentration-effect curve.

Fig. 2. Pretreatment with nitrite increases the concentrations of nitrosylated species (RXNO) in plasma, and enhances total protein and PKC nitrosylation in the vessels. Panels A and B show the concentrations of nitrite and nitrosylated species (RXNO), respectively, in plasma from rats pretreated with oral nitrite (15 mg/kg) or vehicle for five days. Panel C shows a representative SDS/PAGE gel stained with Coomassie Blue to quantify total protein nitrosylation in aortic samples from nitrite or vehicle-treated animals using the SNO-RAC method (“I” corresponds to input: total protein; “o” corresponds to output: nitrosylated protein). The bar graphic shows the quantification of total nitrosylated proteins in the aortic samples. Panel D shows the quantification of PKC expression in the aortas from nitrite or vehicle-treated animals using Western blotting analysis. Panels E show a representative western blotting marking to quantification of PKC nitrosylation in aortic samples from nitrite or vehicle-treated animals. Data are shown as mean ± S.E.M. (n = 5–10/group). *P < 0.05 versus Vehicle. . (For interpretation of the references to colour in this figure legend, the reader is referred to the…

Fig. 3. Pretreatment with S-nitrosogluthatione (GSNO) increases the concentrations of nitrosylated species (RXNO) in plasma, enhances total protein and PKC nitrosylation in the vessels and attenuates the blood pressure responses to angiotensin II in vivo. Panel A shows the increases in mean arterial blood pressure (MAP) after infusion of angiotensin II (0.03, 0.1, 0.3, 1.0 and 3.0 μg/kg, i.v.) in rats pretreated with oral GSNO (0.2 mmol/kg) or vehicle for five days. Panels B and C show the concentrations of nitrite and nitrosylated species (RXNO), respectively, in plasma from rats pretreated with oral GSNO or vehicle. Panel D shows a representative SDS/PAGE gel stained with Coomassie Blue to quantify total protein nitrosylation in aortic samples from GSNO or vehicle-treated animals using the SNO-RAC method (“I” corresponds to input: total protein; “o” corresponds to output: nitrosylated protein). The bar graphic shows the quantification of total nitrosylated proteins in the aortic samples. Panel E shows the quantification of PKC expression in the aortas using Western blotting analysis and show a representative marking to quantify PKC nitrosylation. Panel F show the quantification of PKC…

Fig. 4. Partial thiol depletion prevents oral nitrite from attenuating the blood pressure responses to angiotensin II and blunts oral nitrite-induced increases in plasma concentrations of nitrosylated species, in total protein and PKC nitrosylation in the vessels. Panel A shows the increases in mean arterial blood pressure (MAP) after infusion of angiotensin II (0.03, 0.1, 0.3, 1.0 and 3.0 μg/kg, i.v.) in rats pretreated with the glutathione synthase inhibitor buthionine sulfoximine (BSO) 1.4 mmol/kg every 12 h, i.p., and with oral nitrite (15 mg/kg) or vehicle for five days. BSO pretreatment blunted the effects of oral nitrite, which were found in control animals treated with vehicle instead of BSO and are shown in Fig. 1A). Panels B and C show the concentrations of nitrite and nitrosylated species (RXNO), respectively, in plasma from rats pretreated with vehicle or nitrite, and with BSO or BSO and nitrite for five days. Panel D shows a representative SDS/PAGE gel stained with Coomassie Blue to quantify total protein nitrosylation in aortic samples from the four experimental groups using the SNO-RAC method (“I” corresponds to input: total protein; “o” corresponds to output: nitrosylated…

Fig. 5. Incubation with S-nitrosogluthatione (GSNO) impairs the vascular contraction induced by angiotensin II or by the PKC activator PDBu and this effect is prevented by ascorbate. Panel A shows the vasoconstriction induced by angiotensin II (10−10 to 10−7 mol/l) in aortic ring preparations. The aortic rings were preincubated for 5 min with GSNO 1 mmol/l (or vehicle) in the bath chambers and washed out extensively with modified Krebs salt solution. After eliminating residual GSNO from the bath chambers, the aortic rings were further incubated with ascorbate 5 mmol/l (or vehicle) for additional 15 min and then the concentration-response curves in response to angiotensin II were constructed. Panels B and C show the corresponding pD2 values (the negative logarithm of the concentration producing half maximum effect) and maximum effect, respectively. Panel D shows the vasoconstriction induced by the PKC activator PDBu (10−9 to 10−5 mol/l) in aortic ring preparations preincubated with GSNO (or vehicle) and ascorbate (or vehicle) as described above. Panels E and F show the corresponding pD2 values (the negative logarithm of the concentration producing half maximum effect) and maximum effect,…

Fig. 6. Incubation with sodium nitrite does not affect the vascular contraction induced by angiotensin II or by the PKC activator PDBu. Panel A shows the vasoconstriction induced by angiotensin II (10−10 to 10−7 mol/l) in aortic ring preparations. The aortic rings were preincubated for 5 min with sodium nitrite 1 mmol/l (or vehicle) in the bath chambers and washed out extensively with modified Krebs salt solution. After eliminating residual sodium nitrite from the bath chambers, concentration-response curves in response to angiotensin II were constructed. Panels B and C show the corresponding pD2 values (the negative logarithm of the concentration producing half maximum effect) and maximum effect, respectively. Panel D shows the vasoconstriction induced by the PKC activator PDBu (10−9 to 10−5 mol/l) in aortic ring preparations preincubated with sodium nitrite (or vehicle) as described above. Panels E and F show the corresponding pD2 values (the negative logarithm of the concentration producing half maximum effect) and maximum effect, respectively. Data are shown as mean ± S.E.M. (n = 6/group).

Fig. 7. Incubation of HEK293T cells expressing angiotensin II type 1 receptors (AT1R) with GSNO impairs angiotensin II (but not oxytocin)-stimulated intracellular calcium mobilization. Panel A shows intracellular Ca2+ mobilization in HEK293T cells expressing AT1R and loaded with a Ca2+-sensitive dye and stimulated with angiotensin II (10−12 to 10−6 mol/l) after treatment with GSNO (1 mM) or vehicle for 15 min. Panel B shows control experiments using the same cells expressing oxytocin receptor, which were stimulated with oxytocin (10−12 to 10−7 mol/l) after treatment with GSNO (1 mM) or vehicle for 15 min. Panel C shows a representative SDS/PAGE gel stained with Coomassie Blue to quantify total protein nitrosylation in HEK293T cells (treated with from GSNO or vehicle) using the SNO-RAC method (“I” corresponds to input: total protein; “o” corresponds to output: nitrosylated protein). The bar graphic shows the quantification of total nitrosylated proteins. Data are shown as mean ± S.E.M. (n = 3–4/group). *P < 0.05 versus Vehicle. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)