- Email: [email protected]
P75
S46
Abstracts / Nitric Oxide 31 (2013) S13–S48
determine if mARC can utilize NADH as an electron source in conjunction with these enzymes. Nitric oxide chemiluminescence spectroscopy was used to measure NO-formation rates under anaerobic conditions. Our study established that mARC can generate NO from nitrite in the presence of NADH, cytochrome b5, and cytochrome b5 reductase at pH 7.4. The maximum velocity (Vmax) of NO-formation measured was 5 nmoles NO s 1 mg 1 protein. Moreover, mutation of the putative active site cysteine residue to alanine, and substitution of tungsten for molybdenum, completely abolished enzyme activity. The kinetic data supports our hypothesis and establishes that human mARC is capable of catalyzing reduction of nitrite to NO. Moreover, these data suggest that cysteine 270 and molybdenum are important in the transformation of nitrite to NO. Disclosure: Supported by institution training grant (T32). http://dx.doi.org/10.1016/j.niox.2013.02.076
P75 Dietary inorganic nitrate reduces basal metabolic rate in man Tomas Schiffer, Eddie Weitzberg, Jon O. Lundberg, Filip J. Larsen Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden The integration of the rate-of-living and oxidative damage theory of aging predicts that lifespan extension is linked to low energy metabolism, low reactive oxygen species production rates and a slow aging rate. Recent studies show that inorganic nitrate, an inorganic anion abundant in vegetables, can reduce oxygen consumption during physical exercise and attenuate oxidative stress in animal models of disease. Bioactivation of nitrate involves its active accumulation in saliva and reduction to nitrite by oral bacteria. In a double-blind, randomized cross over designed study we examined the effects of dietary nitrate on resting energy expenditure and markers of oxidative stress in man. Basal metabolic rate (BMR) was measured by indirect calorimetry in 15 young healthy male volunteers after a three day dietary intervention with sodium nitrate (NaNO3, 0.1 mmol/kg/day) or placebo (NaCl). This amount of nitrate corresponds to what is found in 100–300 g of nitrate-rich vegetables such as spinach or beetroot. After the nitrate intervention BMR was 4.3% lower compared to placebo (p < 0.02). The change in BMR correlated strongly to the degree of nitrate accumulation in saliva (r2 = 0.72, p < 0.002). Plasma levels of Malondialdehyde (MDA), a marker of oxidative stress, were lower after nitrate supplementation while thyroid hormone status was unaffected. Vegetables figure prominently in the cuisines of cultures known for their longevity. Future studies will reveal if such life span extension in any way is linked to the high nitrate content of this food group. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.077
P76 H2S and Nitrite anion: Partners in preserving ischemic tissue function Shyamal C. Bir, Gopi K. Kolluru, Xinggui Shen, Christopher B. Pattillo, Christopher G. Kevil Louisiana State University Health Sciences Center, Department of Pathology, Shreveport, LA, United States Background: Hydrogen sulfide (H2S) therapy has been reported to modulate vascular function during ischemia. Although nitric oxide synthase (NOS) dependent mechanisms of H2S mediated protection during tissue ischemia have been reported, little is known regarding the relationship between H2S and nitrite metabolism under similar conditions. Objective: We examined the NOS independent but nitrite dependent molecular mechanisms involved in H2S regulation of NO bioavailability and the importance of these effects during chronic tissue ischemia. Approaches: Permanent unilateral hind limb ischemia was induced in wild type and eNOSKO mice by left femoral artery ligation and excision. Mice were assigned to four different groups (n = 8, each group) and treated with PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S, a H2S donor) twice daily by retro-orbital injection. Hind limb perfusion was determined using laser Doppler flowmetry. Angiogenic and cell proliferation index were detected by the ratio of CD31 to DAPI and Ki67/DAPI positive staining respectively. NO generation by Na2S and nitrite reduction in the presence of xanthine oxidase (XO) in endothelial cells was measured using a NO chemiluminescent analyzer. XO activity, cGMP, and VEGF expression were determined by ELISA. Results: Blood perfusion, angiogenic index, cell proliferation index, and VEGF levels were all significantly increased in mice treated with Na2S compared to PBS control. These effects were blunted by cPTIO treatment both in vivo and in vitro suggesting the involvement of NO in H2S mediated ischemic tissue protection. Na2S and nitrite interaction in hypoxic endothelial cells increased NO production in a XO dependent manner. XO activity was increased in Na2S treated mice compared to PBS control. Both in vitro and in vivo data indicate the involvement of XO mediated nitrite reduction to NO that is dependent on H2S. Na2S therapy also increased cGMP levels, which were still observed in eNOS KO mice. Lastly, VEGF164 aptamer inhibited sulfide induced augmentation of blood flow in mice ischemic tissue indicating VEGF164 as a primary downstream target of H2S mediated nitrite reduction to NO under ischemic conditions. Conclusion: Na2S therapy restores ischemic tissue perfusion through a XO mediated nitrite reduction (NO/cGMP/VEGF) pathway. Thus, H2S mediated NO generation from nitrite would be a novel therapeutic option during ischemic vascular remodeling in the state of metabolic syndrome where NOS dependent pathways are defective. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.078
Abstracts / Nitric Oxide 31 (2013) S13–S48
determine if mARC can utilize NADH as an electron source in conjunction with these enzymes. Nitric oxide chemiluminescence spectroscopy was used to measure NO-formation rates under anaerobic conditions. Our study established that mARC can generate NO from nitrite in the presence of NADH, cytochrome b5, and cytochrome b5 reductase at pH 7.4. The maximum velocity (Vmax) of NO-formation measured was 5 nmoles NO s 1 mg 1 protein. Moreover, mutation of the putative active site cysteine residue to alanine, and substitution of tungsten for molybdenum, completely abolished enzyme activity. The kinetic data supports our hypothesis and establishes that human mARC is capable of catalyzing reduction of nitrite to NO. Moreover, these data suggest that cysteine 270 and molybdenum are important in the transformation of nitrite to NO. Disclosure: Supported by institution training grant (T32). http://dx.doi.org/10.1016/j.niox.2013.02.076
P75 Dietary inorganic nitrate reduces basal metabolic rate in man Tomas Schiffer, Eddie Weitzberg, Jon O. Lundberg, Filip J. Larsen Department of Physiology & Pharmacology, Karolinska Institutet, Stockholm, Sweden The integration of the rate-of-living and oxidative damage theory of aging predicts that lifespan extension is linked to low energy metabolism, low reactive oxygen species production rates and a slow aging rate. Recent studies show that inorganic nitrate, an inorganic anion abundant in vegetables, can reduce oxygen consumption during physical exercise and attenuate oxidative stress in animal models of disease. Bioactivation of nitrate involves its active accumulation in saliva and reduction to nitrite by oral bacteria. In a double-blind, randomized cross over designed study we examined the effects of dietary nitrate on resting energy expenditure and markers of oxidative stress in man. Basal metabolic rate (BMR) was measured by indirect calorimetry in 15 young healthy male volunteers after a three day dietary intervention with sodium nitrate (NaNO3, 0.1 mmol/kg/day) or placebo (NaCl). This amount of nitrate corresponds to what is found in 100–300 g of nitrate-rich vegetables such as spinach or beetroot. After the nitrate intervention BMR was 4.3% lower compared to placebo (p < 0.02). The change in BMR correlated strongly to the degree of nitrate accumulation in saliva (r2 = 0.72, p < 0.002). Plasma levels of Malondialdehyde (MDA), a marker of oxidative stress, were lower after nitrate supplementation while thyroid hormone status was unaffected. Vegetables figure prominently in the cuisines of cultures known for their longevity. Future studies will reveal if such life span extension in any way is linked to the high nitrate content of this food group. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.077
P76 H2S and Nitrite anion: Partners in preserving ischemic tissue function Shyamal C. Bir, Gopi K. Kolluru, Xinggui Shen, Christopher B. Pattillo, Christopher G. Kevil Louisiana State University Health Sciences Center, Department of Pathology, Shreveport, LA, United States Background: Hydrogen sulfide (H2S) therapy has been reported to modulate vascular function during ischemia. Although nitric oxide synthase (NOS) dependent mechanisms of H2S mediated protection during tissue ischemia have been reported, little is known regarding the relationship between H2S and nitrite metabolism under similar conditions. Objective: We examined the NOS independent but nitrite dependent molecular mechanisms involved in H2S regulation of NO bioavailability and the importance of these effects during chronic tissue ischemia. Approaches: Permanent unilateral hind limb ischemia was induced in wild type and eNOSKO mice by left femoral artery ligation and excision. Mice were assigned to four different groups (n = 8, each group) and treated with PBS, 0.1, 0.5 and 1 mg/kg sodium sulfide (Na2S, a H2S donor) twice daily by retro-orbital injection. Hind limb perfusion was determined using laser Doppler flowmetry. Angiogenic and cell proliferation index were detected by the ratio of CD31 to DAPI and Ki67/DAPI positive staining respectively. NO generation by Na2S and nitrite reduction in the presence of xanthine oxidase (XO) in endothelial cells was measured using a NO chemiluminescent analyzer. XO activity, cGMP, and VEGF expression were determined by ELISA. Results: Blood perfusion, angiogenic index, cell proliferation index, and VEGF levels were all significantly increased in mice treated with Na2S compared to PBS control. These effects were blunted by cPTIO treatment both in vivo and in vitro suggesting the involvement of NO in H2S mediated ischemic tissue protection. Na2S and nitrite interaction in hypoxic endothelial cells increased NO production in a XO dependent manner. XO activity was increased in Na2S treated mice compared to PBS control. Both in vitro and in vivo data indicate the involvement of XO mediated nitrite reduction to NO that is dependent on H2S. Na2S therapy also increased cGMP levels, which were still observed in eNOS KO mice. Lastly, VEGF164 aptamer inhibited sulfide induced augmentation of blood flow in mice ischemic tissue indicating VEGF164 as a primary downstream target of H2S mediated nitrite reduction to NO under ischemic conditions. Conclusion: Na2S therapy restores ischemic tissue perfusion through a XO mediated nitrite reduction (NO/cGMP/VEGF) pathway. Thus, H2S mediated NO generation from nitrite would be a novel therapeutic option during ischemic vascular remodeling in the state of metabolic syndrome where NOS dependent pathways are defective. Disclosure: Nothing to disclose. http://dx.doi.org/10.1016/j.niox.2013.02.078