Alexandra B. Milsom

Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans. Role for Nitrite-Derived NO

Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure–lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO3 capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO3 (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.

Hypoxic Modulation of Exogenous Nitrite-Induced Vasodilation in Humans

Background— It has been proposed that under hypoxic conditions, nitrite may release nitric oxide, which causes potent vasodilation. We hypothesized that nitrite would have a greater dilator effect in capacitance than in resistance vessels because of lower oxygen tension and that resistance-vessel dilation should become more pronounced during hypoxemia. The effect of intra-arterial infusion of nitrite on forearm blood flow and forearm venous volumes was assessed during normoxia and hypoxia. Methods and Results— Forty healthy volunteers were studied. After baseline infusion of 0.9% saline, sodium nitrite was infused at incremental doses from 40 nmol/min to 7.84 &mgr;mol/min. At each stage, forearm blood flow was measured by strain-gauge plethysmography. Forearm venous volume was assessed by radionuclide plethysmography. Changes in forearm blood flow and forearm venous volume in the infused arm were corrected for those in the control arm. The peak percentage of venodilation during normoxia was 35.8±3.4% (mean±SEM) at 7.84 &mgr;mol/min (P<0.001) and was similar during hypoxia. In normoxia, arterial blood flow, assessed by the forearm blood flow ratio, increased from 1.04±0.09 (baseline) to 1.62±0.18 (nitrite; P<0.05) versus 1.07±0.09 (baseline) to 2.37±0.15 (nitrite; P<0.005) during hypoxia. This result was recapitulated in vitro in vascular rings. Conclusions— Nitrite is a potent venodilator in normoxia and hypoxia. Arteries are modestly affected in normoxia but potently dilated in hypoxia, which suggests the important phenomenon of hypoxic augmentation of nitrite-mediated vasodilation in vivo. The use of nitrite as a selective arterial vasodilator in ischemic territories and as a potent venodilator in heart failure has therapeutic implications.

Mechanisms Underlying Erythrocyte and Endothelial Nitrite Reduction to Nitric Oxide in Hypoxia: Role for Xanthine Oxidoreductase and Endothelial Nitric Oxide Synthase

Reduction of nitrite (NO2−) provides a major source of nitric oxide (NO) in the circulation, especially in hypoxemic conditions. Our previous studies suggest that xanthine oxidoreductase (XOR) is an important nitrite reductase in the heart and kidney. Herein, we have demonstrated that conversion of nitrite to NO by blood vessels and RBCs was enhanced in the presence of the XOR substrate xanthine (10 &mgr;mol/L) and attenuated by the XOR inhibitor allopurinol (100 &mgr;mol/L) in acidic and hypoxic conditions only. Whereas endothelial nitric oxide synthase (eNOS) inhibition had no effect on vascular nitrite reductase activity, in RBCs L-NAME, L-NMMA, and l-arginine inhibited nitrite-derived NO production by >50% (P<0.01) at pH 7.4 and 6.8 under hypoxic conditions. Western blot and immunohistochemical analysis of RBC membranes confirmed the presence of eNOS and abundant XOR on whole RBCs. Thus, XOR and eNOS are ideally situated on the membranes of RBCs and blood vessels to generate intravascular vasodilator NO from nitrite during ischemic episodes. In addition to the proposed role of deoxyhemoglobin, our findings suggest that the nitrite reductase activity within the circulation, under hypoxic conditions (at physiological pH), is mediated by eNOS; however, as acidosis develops, a substantial role for XOR becomes evident.

Vasorelaxation by Red Blood Cells and Impairment in Diabetes Reduced Nitric Oxide and Oxygen Delivery by Glycated Hemoglobin

Abstract— Vascular dysfunction in diabetes is attributed to lack of bioavailable nitric oxide (NO) and is postulated as a primary cause of small vessel complications as a result of poor glycemic control. Although it has been proposed that NO is bound by red blood cells (RBCs) and can induce relaxation of blood vessels distal to its site of production in the normal circulation, the effect of RBC glycation on NO binding and relaxation of hypoxic vessels is unknown. We confirm RBC-induced vessel relaxation is inversely related to tissue oxygenation and is proportional to RBC S-nitrosohemoglobin (HbSNO) content (but not nitrosylhemoglobin content). We show more total NO bound inside highly glycated RBCs (0.0134 versus 0.0119 NO/Hb, respectively; P <0.05) although proportionally less HbSNO (0.0053 versus 0.0088 NO/Hb, respectively; P <0.05). We also show glycosylation impairs the vasodilator function of RBCs within a physiological range of tissue oxygenation. These findings may represent an important contribution to reduced NO bioavailability in the microvasculature in diabetes.

Enhanced Vasodilator Activity of Nitrite in Hypertension Critical Role for Erythrocytic Xanthine Oxidoreductase and Translational Potential

Elevation of circulating nitrite (NO2 −) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naïve grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold (P<0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures (P<0.001) and pulse wave velocity (P<0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy.Elevation of circulating nitrite (NO2−) levels causes vasodilatation and lowers blood pressure in healthy volunteers. Whether these effects and the underpinning mechanisms persist in hypertension is unknown. Therefore, we investigated the consequences of systemic nitrite elevation in spontaneously hypertensive rats and conducted proof-of-principle studies in patients. Nitrite caused dose-dependent blood pressure–lowering that was profoundly enhanced in spontaneously hypertensive rats versus normotensive Wistar Kyoto controls. This effect was virtually abolished by the xanthine oxidoreductase (XOR) inhibitor, allopurinol, and associated with hypertension-specific XOR-dependent nitrite reductase activity localized to the erythrocyte but not the blood vessel wall. To determine whether these pathways translate to human hypertension, we investigated the effects of elevation of circulating nitrite levels in 15 drug naive grade 1 hypertensives. To elevate nitrite, we used a dose of dietary nitrate (≈3.5 mmol) that elevated nitrite levels ≈1.5-fold ( P <0.01); a rise shown previously to exert no significant blood pressure–lowering effects in normotensives. This dose caused substantial reductions in systolic (≈12 mm Hg) and diastolic blood pressures ( P <0.001) and pulse wave velocity ( P <0.05); effects associated with elevations in erythrocytic XOR expression and XOR-dependent nitrite reductase activity. Our observations demonstrate the improved efficacy of inorganic nitrate and nitrite in hypertension as a consequence of increased erythrocytic XOR nitrite reductase activity and support the concept of dietary nitrate supplementation as an effective, but simple and inexpensive, antihypertensive strategy. # Novelty and Significance {#article-title-61}

Dietary nitrate ameliorates pulmonary hypertension: cytoprotective role for endothelial nitric oxide synthase and xanthine oxidoreductase

Background— Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO3−) and nitrite (NO2−) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods and Results— Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol. Conclusions— These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH. # Clinical Perspective {#article-title-52}Background— Pulmonary hypertension (PH) is a multifactorial disease characterized by increased pulmonary vascular resistance and right ventricular failure; morbidity and mortality remain unacceptably high. Loss of nitric oxide (NO) bioactivity is thought to contribute to the pathogenesis of PH, and agents that augment pulmonary NO signaling are clinically effective in the disease. Inorganic nitrate (NO3 −) and nitrite (NO2 −) elicit a reduction in systemic blood pressure in healthy individuals; this effect is underpinned by endogenous and sequential reduction to NO. Herein, we determined whether dietary nitrate and nitrite might be preferentially reduced to NO by the hypoxia associated with PH, and thereby offer a convenient, inexpensive method of supplementing NO functionality to reduce disease severity. Methods and Results— Dietary nitrate reduced the right ventricular pressure and hypertrophy, and pulmonary vascular remodeling in wild-type mice exposed to 3 weeks of hypoxia; this beneficial activity was mirrored largely by dietary nitrite. The cytoprotective effects of dietary nitrate were associated with increased plasma and lung concentrations of nitrite and cGMP. The beneficial effects of dietary nitrate and nitrite were reduced in mice lacking endothelial NO synthase or treated with the xanthine oxidoreductase inhibitor allopurinol. Conclusions— These data demonstrate that dietary nitrate, and to a lesser extent dietary nitrite, elicit pulmonary dilatation, prevent pulmonary vascular remodeling, and reduce the right ventricular hypertrophy characteristic of PH. This favorable pharmacodynamic profile depends on endothelial NO synthase and xanthine oxidoreductase -catalyzed reduction of nitrite to NO. Exploitation of this mechanism (ie, dietary nitrate/nitrite supplementation) represents a viable, orally active therapy for PH.

Role for endothelial nitric oxide synthase in nitrite-induced protection against renal ischemia–reperfusion injury in mice

Nitrite is protective against renal ischemia/reperfusion injury (IRI); an effect due to its reduction to nitric oxide (NO). In addition to other reductase pathways, endothelial NO synthase (eNOS) may also facilitate nitrite reduction in ischemic environments. We investigated the role of eNOS in sodium nitrite (60 microM, 10 ml/kg applied topically 1 min before reperfusion)-induced protection against renal IRI in C57/BL6 wild-type (WT) and eNOS knockout (eNOS KO) mice subjected to bilateral renal ischemia (30 min) and reperfusion (24h). Markers of renal dysfunction (plasma [creatinine] and [urea]), damage (tubular histology) and inflammation (cell recruitment) were elevated following IRI in WT mice; effects significantly reduced following nitrite treatment. Chemiluminescence analysis of cortical and medullary sections of the kidney demonstrated rapid (within 1 min) distribution of nitrite following application. Whilst IRI caused a significant (albeit substantially reduced compared to WT mice) elevation of markers of renal dysfunction and damage in eNOS KO mice, the beneficial effects of nitrite were absent or reduced, respectively. Moreover, nitrite treatment enhanced renal dysfunction in the form of increased plasma [creatinine] in eNOS KO mice. Confirmation of nitrite reductase activity of eNOS was provided by demonstration of nitrite (100 microM)-derived NO production by kidney homogenates of WT mice, that was significantly reduced by L-NMMA. L-NMMA was without effect using kidney homogenates of eNOS KO mice. These results support a role for eNOS in the pathways activated during renal IRI and also identify eNOS as a nitrite reductase in ischemic conditions; activity which in part underlies the protective effects of nitrite.

On the dynamics of nitrite, nitrate and other biomarkers of nitric oxide production in inflammatory bowel disease

Nitrite and nitrate are frequently used surrogate markers of nitric oxide (NO) production. Using rat models of acute and chronic DSS-induced colitis we examined the applicability of these and other NO-related metabolites, in tissues and blood, for the characterization of inflammatory bowel disease. Global NO dynamics were assessed by simultaneous quantification of nitrite, nitrate, nitroso and nitrosyl species over time in multiple compartments. NO metabolite levels were compared to a composite disease activity index (DAI) and contrasted with measurements of platelet aggregability, ascorbate redox status and the effects of 5-aminosalicylic acid (5-ASA). Nitroso products in the colon and in other organs responded in a manner consistent with the DAI. In contrast, nitrite and nitrate, in both intra- and extravascular compartments, exhibited variations that were not always in step with the DAI. Extravascular nitrite, in particular, demonstrated significant temporal instabilities, ranging from systemic drops to marked increases. The latter was particularly evident after cessation of the inflammatory stimulus and accompanied by profound ascorbate oxidation. Treatment with 5-ASA effectively reversed these fluctuations and the associated oxidative and nitrosative stress. Platelet activation was enhanced in both the acute and chronic model. Our results offer a first glimpse into the systemic nature of DSS-induced inflammation and reveal a greater complexity of NO metabolism than previously envisioned, with a clear dissociation of nitrite from other markers of NO production. The remarkable effectiveness of 5-ASA to abrogate the observed pattern of nitrite instability suggests a hitherto unrecognized role of this molecule in either development or resolution of inflammation. Its possible link to tissue oxygen consumption and the hypoxia that tends to accompany the inflammatory process warrants further investigation.

Alterations in nitric oxide and endothelin-1 bioactivity underlie cerebrovascular dysfunction in ApoE-deficient mice

Hypercholesterolemia is associated with decreased nitric oxide (NO) bioavailability and endothelial dysfunction, a phenomenon thought to have a major role in the altered cerebral blood flow evident in stroke. Therefore, strategies that increase endothelial NO production have potential utility. Vascular reactivity of the middle cerebral artery (MCA) from C57BL/6J wild-type (WT) mice, apolipoprotein-E knockout (ApoE−/−) mice, and mice treated with the phosphodiesterase inhibitor cilostazol (100 mg/kg) was analyzed using the tension myograph. Contractile responses to endothelin-1 were significantly enhanced in MCA from ApoE−/− mice compared with WT mice (P<0.01), an effect absent in cilostazol-treated ApoE−/− mice. Acetylcholine-induced relaxation (which is entirely NO-dependent) was significantly impaired in MCA of ApoE−/− mice compared with WT mice (P<0.05), again an effect prevented by cilostazol treatment. Endothelial NOS phosphorylation at Ser1179 was decreased in the aorta of ApoE−/− mice compared with WT mice (P<0.05), an effect normalized by cilostazol. Taken together, our data suggest that the endothelial dysfunction observed in MCA associated with hypercholesterolemia is prevented by cilostazol, an effect likely due to the increase in eNOS phosphorylation and, therefore, activity.

Contributions of Nitric Oxide Synthases, Dietary Nitrite/Nitrate, and Other Sources to the Formation of NO Signaling Products

UNLABELLED Mice lacking all three nitric oxide synthase (NOS) genes remain viable even though deletion of the major downstream target of NO, soluble guanylyl cyclase, is associated with a dramatically shortened life expectancy. Moreover, findings of relatively normal flow responses in eNOS knockouts are generally attributed to compensatory mechanisms including upregulation of remaining NOS isoforms, but the alternative possibility that dietary nitrite/nitrate (NOx) may contribute to basal levels of NO signaling has never been investigated. AIM The aim of the present study was to examine how NO signaling products (nitrosated and nitrosylated proteins) and NO metabolites (nitrite, nitrate) are affected by single NOS deletions and whether dietary NOx plays a compensatory role in any deficiency. Specifically, we sought to ascertain whether profound alterations of these products arise upon genetic deletion of either NOS isoform, inhibition of all NOS activity, NOx restriction, or all of the above. RESULTS Our results indicate that while some significant changes do indeed occur, they are surprisingly moderate and compartmentalized to specific tissues. Unexpectedly, even after pharmacological inhibition of all NOSs and restriction of dietary NOx intake in eNOS knockout mice significant levels of NO-related products remain. Innovation/Conclusion: These findings suggest that a yet unidentified source of NO, unrelated to NOSs or dietary NOx, may be sustaining basal NO signaling in tissues. Given the significance of NO for redox regulation in health and disease, it would seem to be important to identify the nature of this additional source of NO products as it may offer new therapeutic avenues for correcting NO deficiencies.