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IntroductionNitrate supplementation in the form of beetroot juice (BRJ) ingestion has been shown to improve exercise tolerance during acute hypoxia, but its effect on exercise physiology remains unstudied during sustained terrestrial high altitude exposure. We hypothesized that performing exercise at high altitude would lower circulating nitrate and nitrite levels and that BRJ ingestion would reverse this phenomenon while concomitantly improving key determinants of aerobic exercise performance.MethodsTwenty seven healthy volunteers (21 male) underwent a series of exercise tests at sea level (SL, London, 75 m) and again after 5–8 days at high altitude (HA, Capanna Regina Margherita or “Margherita Hut,” 4,559 m). Using a double-blind protocol, participants were randomized to consume a beetroot/fruit juice beverage (three doses per day) with high levels of nitrate (∼0.18 mmol/kg/day) or a nitrate-depleted placebo (∼11.5 μmoles/kg/day) control drink, from 3 days prior to the exercise trials until completion. Submaximal constant work rate cycle tests were performed to determine exercise efficiency and a maximal incremental ramp exercise test was undertaken to measure aerobic capacity, using breath-by-breath pulmonary gas exchange measurements throughout. Concentrations of nitrate, nitrite and nitrosation products were quantified in plasma samples collected at 5 timepoints during the constant work rate tests. Linear mixed modeling was used to analyze data.ResultsAt both SL and HA, plasma nitrate concentrations were elevated in the nitrate supplementation group compared to placebo (P < 0.001) but did not change throughout increasing exercise work rate. Delta exercise efficiency was not altered by altitude exposure (P = 0.072) or nitrate supplementation (P = 0.836). V̇O2peak decreased by 24% at high altitude (P < 0.001) and was lower in the nitrate-supplemented group at both sea level and high altitude compared to placebo (P = 0.041). Dietary nitrate supplementation did not alter other peak exercise variables or oxygen consumption at anaerobic threshold. Circulating nitrite and S-nitrosothiol levels unexpectedly rose in a few individuals right after cessation of exercise at high altitude.ConclusionWhilst regularly consumed during an 8 days expedition to terrestrial high altitude, nitrate supplementation did not alter exercise efficiency and other exercise physiological variables, except decreasing V̇O2peak. These results and those of others question the practical utility of BRJ consumption during prolonged altitude exposure.

Acute mountain sickness (AMS) occurs when there is failure of acclimatisation to high altitude. The aim of this study was to describe the relationship between physiological variables and the incidence of AMS during ascent to 5300 m. A total of 332 lowland‐dwelling volunteers followed an identical ascent profile on staggered treks. Self‐reported symptoms of AMS were recorded daily using the Lake Louise score (mild 3–4; moderate‐severe ≥5), alongside measurements of physiological variables (heart rate, respiratory rate (RR), peripheral oxygen saturation (SpO2) and blood pressure) before and after a standardised Xtreme Everest Step‐Test (XEST). The overall occurrence of AMS among participants was 73.5% (23.2% mild, 50.3% moderate–severe). There was no difference in gender, age, previous AMS, weight or body mass index between participants who developed AMS and those who did not. Participants who had not previously ascended >5000 m were more likely to get moderate‐to‐severe AMS. Participants who suffered moderate‐to‐severe AMS had a lower resting SpO2 at 3500 m (88.5 vs. 89.6%, p = 0.02), while participants who suffered mild or moderate‐to‐severe AMS had a lower end‐exercise SpO2 at 3500 m (82.2 vs. 83.8%, p = 0.027; 81.5 vs. 83.8%, p 5000 m (OR 2.740, p‐value 0.003) predicted the development of moderate‐to‐severe AMS. The Xtreme Everest Step‐Test offers a simple, reproducible field test to help predict AMS, albeit with relatively limited predictive precision., We evaluated the performance of a simple exercise challenge at altitude (the Xtreme Everest Step‐Test) in predicting acute mountain sickness (AMS) on a trek to Everest Base Camp (5300 m). The overall occurrence of AMS during the trek was high (73.5%). Lower peripheral oxygen saturation (SpO2) following the Xtreme Everest Step‐Test at 3500 m and no previous exposure to altitude >5000 m predicted the development of moderate to severe AMS (Lake Louise score ≥5) during the trek. The Xtreme Everest Step‐Test offers a simple, reproducible field test to contribute towards the prediction of AMS, albeit with relatively limited predictive precision.

A large yet heterogeneous body of literature exists suggesting that endothelial dysfunction appears early in type 1 diabetes, due to hyperglycemia-induced oxidative stress. The latter may also affect vascular smooth muscles (VSM) function, a layer albeit less frequently considered in that pathology. This meta-analysis aims at evaluating the extent, and the contributing risk factors, of early endothelial dysfunction, and of the possible concomitant VSM dysfunction, in type 1 diabetes. PubMed, Web of Sciences, Cochrane Library databases were screened from their respective inceptions until October 2019. We included studies comparing vasodilatory capacity depending or not on endothelium (i.e., endothelial function or VSM function, respectively) in patients with uncomplicated type 1 diabetes and healthy controls. Fifty-eight articles studying endothelium-dependent function, among which 21 studies also assessed VSM, were included. Global analyses revealed an impairment of standardized mean difference (SMD) (Cohen's d) of endothelial function: -0.61 (95% CI: -0.79, -0.44) but also of VSM SMD: -0.32 (95% CI: -0.57, -0.07). The type of stimuli used (i.e., exercise, occlusion-reperfusion, pharmacological substances, heat) did not influence the impairment of the vasodilatory capacity. Endothelial dysfunction appeared more pronounced within macrovascular than microvascular beds. The latter was particularly altered in cases of poor glycemic control [HbA > 67 mmol/mol (8.3%)]. This meta-analysis not only corroborates the presence of an early impairment of endothelial function, even in response to physiological stimuli like exercise, but also highlights a VSM dysfunction in children and adults with type 1 diabetes. Endothelial dysfunction seems to be more pronounced in large than small vessels, fostering the debate on their relative temporal appearance. 11

Lamia Heikal,1 Pietro Ghezzi,1 Manuela Mengozzi,1 Blanka Stelmaszczuk,2 Martin Feelisch,2 Gordon AA Ferns1 1Brighton and Sussex Medical School, Falmer, Brighton, 2Clinical and Experimental Sciences, Faculty of Medicine, University of Southampton, Southampton General Hospital and Institute for Life Sciences, Southampton, UK Abstract: The cytoprotective effects of erythropoietin (EPO) and an EPO-related nonerythropoietic analog, pyroglutamate helix B surface peptide (pHBSP), were investigated in an in vitro model of bovine aortic endothelial cell injury under normoxic (21% O2) and hypoxic (1% O2) conditions. The potential molecular mechanisms of these effects were also explored. Using a model of endothelial injury (the scratch assay), we found that, under hypoxic conditions, EPO and pHBSP enhanced scratch closure by promoting cell migration and proliferation, but did not show any effect under normoxic conditions. Furthermore, EPO protected bovine aortic endothelial cells from staurosporine-induced apoptosis under hypoxic conditions. The priming effect of hypoxia was associated with stabilization of hypoxia inducible factor-1α, EPO receptor upregulation, and decreased Ser-1177 phosphorylation of endothelial nitric oxide synthase (NOS); the effect of hypoxia on the latter was rescued by EPO. Hypoxia was associated with a reduction in nitric oxide (NO) production as assessed by its oxidation products, nitrite and nitrate, consistent with the oxygen requirement for endogenous production of NO by endothelial NOS. However, while EPO did not affect NO formation in normoxia, it markedly increased NO production, in a manner sensitive to NOS inhibition, under hypoxic conditions. These data are consistent with the notion that the tissue-protective actions of EPO-related cytokines in pathophysiological settings associated with poor oxygenation are mediated by NO. These findings may be particularly relevant to atherogenesis and postangioplasty restenosis. Keywords: erythropoietin, pyroglutamate helix B surface peptide, scratch assay, proliferation, migration, apoptosis

Graphical abstract, Nitrate (NO3−)-rich foods such as green leafy vegetables are not only part of a healthy diet, but increasingly marketed for primary prevention of cardiovascular disease (CVD) and used as ergogenic aids by competitive athletes. While there is abundant evidence for mild hypotensive effects of nitrate on acute application there is limited data on chronic intake in humans, and results from animal studies suggest no long-term benefit. This is important as nitrate can also promote the formation of nitrosamines. It is therefore classified as ‘probably carcinogenic to humans', although a beneficial effect on CVD risk might compensate for an increased cancer risk. Dietary nitrate requires reduction to nitrite (NO2−) by oral commensal bacteria to contribute to the formation of nitric oxide (NO). The extensive crosstalk between NO and hydrogen sulfide (H2S) related metabolites may further affect nitrate’s bioactivity. Using nitrate and nitrite concentrations of drinking water − the only dietary source continuously monitored for which detailed data exist − in conjunction with data of >14,000 participants of the EPIC-Norfolk study, we found no inverse associations with blood pressure or CVD risk. Instead, we found a strong interaction with sulfate (SO42−). At low sulfate concentrations, nitrate was inversely associated with BP (−4 mmHg in top quintile) whereas this was reversed at higher concentrations (+3 mmHg in top quintile). Our findings have a potentially significant impact for pharmacology, physiology and public health, redirecting our attention from the oral microbiome and mouthwash use to interaction with sulfur-containing dietary constituents. These results also indicate that nitrate bioactivation is more complex than hitherto assumed. The modulation of nitrate bioactivity by sulfate may render dietary lifestyle interventions aimed at increasing nitrate intake ineffective and even reverse potential antihypertensive effects, warranting further investigation.

Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases., This paper reports the first example of targeted anti-biofilm therapy in human disease. We have demonstrated that using low-dose nitric oxide as a non-bactericidal signaling molecule to induce biofilm dispersal may be useful as a novel adjunctive therapy to treat chronic pseudomonal biofilm infection in cystic fibrosis.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Bacterial biofilms show high tolerance towards antibiotics and are a significant problem in clinical settings where they are a primary cause of chronic infections. Novel therapeutic strategies are needed to improve anti-biofilm efficacy and support reduction in antibiotic use. Treatment with exogenous nitric oxide (NO) has been shown to modulate bacterial signaling and metabolic processes that render biofilms more susceptible to antibiotics.We previously reported on cephalosporin-3’-diazeniumdiolates (C3Ds) as NO-donor prodrugs designed to selectively deliver NO to bacterial infection sites following reaction with b-lactamases. With structures based on cephalosporins, C3Ds could, in principal, also be triggered to release NO following b-lactam cleavage mediated by transpeptidases/penicillin-binding proteins (PBPs), the antibacterial target of cephalosporin antibiotics. Transpeptidase-reactive C3Ds could potentially show both NO-mediated anti-biofilm properties and intrinsic (b-lactam-mediated) antibacterial effects. This dual-activity concept was explored using Streptococcus pneumoniae, a species that lacks b-lactamases but relies on transpeptidases for cell-wall synthesis. Treatment with PYRRO-C3D (a representative C3D containing the diazeniumdiolate NO donor PYRRO-NO) was found to significantly reduce viability of planktonic and biofilm pneumococci, demonstrating that C3Ds can elicit direct, cephalosporin-like antibacterial activity in the absence of b-lactamases. While NO release from PYRRO-C3D in the presence of pneumococci was confirmed, the anti-pneumococcal action of the compound was shown to arise exclusively from the b-lactam component and not through NO-mediated effects. The compound showed similar potency to amoxicillin against S. pneumoniae biofilms and greater efficacy than azithromycin, highlighting the potential of C3Ds as new agents for treating pneumococcal infections.

Exercise is an effective intervention for prevention and treatment of type 2 diabetes. Skeletal muscle combines multiple signals contributing to the beneficial effects of exercise on cardiometabolic health. Inorganic nitrate increases exercise efficiency, tolerance and performance. The transcriptional regulator peroxisome proliferator-activated receptorγ coactivator1α (PGC1α) coordinates the exercise-stimulated skeletal muscle fiber-type switch from glycolytic fast-twitch (type IIb) to oxidative slow-twitch (type I) and intermediate (type IIa) fibers; an effect reversed in insulin resistance and diabetes. We find that nitrate induces PGC1α expression and a switch towards type I and IIa fibers in rat muscle and myotubes in vitro Nitrate induces the release of exercise/PGC1α-dependent myokine FNDC5/irisin, and β-aminoisobutyric acid from myotubes, and muscle in rats and humans. Both exercise and nitrate stimulated, PGC1α-mediated, γ-aminobutyric acid (GABA) secretion from muscle. Circulating GABA concentrations were increased in exercising mice and nitrate-treated rats and humans, thus, GABA may function as an exercise/PGC1α-mediated myokine-like small molecule. Moreover, nitrate increased circulating growth hormone levels in humans and rodents. Nitrate induces physiological responses that mimic exercise training and may underlie the beneficial effects of this metabolite on exercise and cardiometabolic health.

Although nitrite (NO(2)(-)) and nitrate (NO(3)(-)) have been considered traditionally inert byproducts of nitric oxide (NO) metabolism, recent studies indicate that NO(2)(-) represents an important source of NO for processes ranging from angiogenesis through hypoxic vasodilation to ischemic organ protection. Despite intense investigation, the mechanisms through which NO(2)(-) exerts its physiological/pharmacological effects remain incompletely understood. We sought to systematically investigate the fate of NO(2)(-) in hypoxia from cellular uptake in vitro to tissue utilization in vivo using the Wistar rat as a mammalian model. We find that most tissues (except erythrocytes) produce free NO at rates that are maximal under hypoxia and that correlate robustly with each tissue's capacity for mitochondrial oxygen consumption. By comparing the kinetics of NO release before and after ferricyanide addition in tissue homogenates to mathematical models of NO(2)(-) reduction/NO scavenging, we show that the amount of nitrosylated products formed greatly exceeds what can be accounted for by NO trapping. This difference suggests that such products are formed directly from NO(2)(-), without passing through the intermediacy of free NO. Inhibitor and subcellular fractionation studies indicate that NO(2)(-) reductase activity involves multiple redundant enzymatic systems (i.e. heme, iron-sulfur cluster, and molybdenum-based reductases) distributed throughout different cellular compartments and acting in concert to elicit NO signaling. These observations hint at conserved roles for the NO(2)(-)-NO pool in cellular processes such as oxygen-sensing and oxygen-dependent modulation of intermediary metabolism.

Background Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. Results Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα−/− mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. Conclusions Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle. Electronic supplementary material The online version of this article (doi:10.1186/s12915-015-0221-6) contains supplementary material, which is available to authorized users.

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO?), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO? is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO? synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Because nitric oxide (NO) inhibits the expression of endothelial leukocyte adhesion molecules, NO-generating compounds have major therapeutic potential for use outside their classical indications. We report on the in vitro potential antiatherogenicity of two novel cysteine-containing NO donors, SP/W 3672, a fast spontaneous NO releaser, and its prodrug SP/W 5186, which liberates NO after bioactivation. The ability of these two compounds to inhibit monocyte adhesion and surface expression of endothelial adhesion molecules was evaluated and compared with that of other NO donors. SP/W 5186 and SP/W 3672 inhibited the adhesion of U937 monocytes to cultured human endothelial cells more potently than S-nitrosoglutathione (GSNO) or spermine NONOate, whereas nitroglycerin and isosorbide dinitrate were ineffective at comparable concentrations. A similar rank order of potency was found for the inhibition of expression of the adhesion molecules vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin as well as for major histocompatibility complex class II antigen expression. Estimated IC(50) values for vascular cell adhesion molecule-1 were400 microM for SP/W 4744 (control for SP/W 3672 lacking the cysteine moiety), 200 microM for GSNO and spermine NONOate, 80 microM for SP/W 3672, and 50 microM for SP/W 5186. Moreover, SP/W 5186 inhibited VCAM-1 mRNA levels more potently than GSNO. This effect was likely to be transcriptional because mRNA degradation was not affected. In conclusion, SP/W 3672 and SP/W 5186 are novel potent inhibitors of endothelial activation, and this effect appears to relate to their ability to liberate NO for prolonged periods of time, either spontaneously or after conversion to active hydrolytic products.

This study was undertaken to investigate the possible involvement of K+ channels in PGE1-mediated vasodilatation. The increase in coronary flow elicited by PGE1 in isolated working rat hearts was attenuated by phentolamine and glibenclamide, inhibitors of ATP-regulated K+ channels, whereas apamin and charybdotoxin, inhibitors of calcium-activated K+ channels, were ineffective. In the anaesthetized rat, the duration of the hypotensive action of PGE1 was markedly attenuated by glibenclamide. It is concluded that the vasodilatory action of PGE1 in the coronary and systemic circulation of the rat is, at least in part, mediated via an opening of ATP-sensitive K+ channels.

Nitrovasodilators are prodrugs which, although chemically heterogenous, exert their pharmacodynamic action via a common pathway, i.e. the release of nitric oxide (NO). The NO, which results from metabolism of nitrovasodilators in vascular and non-vascular cells, stimulates the cytosolic enzyme guanylyl cyclase leading to an increase in the concentration of intracellular cyclic guanosine monophosphate (cGMP). In general, the rate of NO generation from the individual compounds correlates well with the extent of cGMP increase and their potency to relax vascular tissue. The amounts of NO generated are sufficient to inhibit platelet aggregation and to induce disaggregation. Nitrovasodilators thus mimic the action of endothelium-derived relaxing factor (EDRF). After more than a century of empiric use, the application of nitrovasodilators today may be regarded as causal therapy, since these drugs act by substituting an endogenous factor, the production or release of which is impaired under pathophysiological circumstances associated with endothelial dysfunction. Marked differences exist between individual compound classes with regard to bioactivation mechanisms, cofactor requirements, and the extent and nature of the concomittant formation of metabolites other than NO. This review describes the discovery of the mode of action of nitrovasodilators and our current understanding of the pathways involved in their bioactivation and biodegradation with special emphasis on the enzymatic and non-enzymatic metabolism of organic nitrates. In addition, the in-vivo metabolism of NO is reviewed briefly