Increased Nitric Oxide Availability Research Articles

Dietary nitrate flows and accumulation in pig organs:15N-tracer study

Nitric oxide (NO) is a free radical with multiple functions in virtually all cells. In mammals, NO is generated by nitric oxide synthase from arginine and by reduction of nitrate to nitrite by tissue xanthine oxidoreductase (XOR) and microbiome. Nitrite is then available for reduction to NO by XOR or several heme proteins. Previously we reported that 1) skeletal muscle acts as a body nitrate reservoir, and 2) stored nitrate is systemically, as well as locally used to generate nitrite and NO. Recently, we identified two additional nitrate storage organs - skeleton and skin. At baseline conditions, nitrate concentration in skeleton was 96±63 nmol/g, skeletal muscle 57±39 nmol/g and skin 56±22 nmol/g. For comparison, plasma and liver contained 34±19 nmol/g and 15±5 nmol/g of nitrate, respectively. We used 15N-labeled dose of dietary nitrate to trace its distribution among organs in time. Three hours after nitrate bolus ingestion, total nitrate levels significantly increased in all organs, reaching 221±91 nmol/g in plasma, 126±55 nmol/g in skin, 119±38 nmol/g in skeleton, 100±79 mmol/g in skeletal muscle, and 21±5 nmol/g in liver. As shown by MS, 15N-labeled nitrate distributes in non-uniform way, accumulating largely in skin. As expected, nitrate reductase activity is highest in liver, but also substantial in skin and skeletal muscle, as seen by presence of 15N-labeled nitrite. With consideration of skeletal muscle, skin and skeleton as three largest organs in mammals, these results underline their major role in maintaining homeostasis of the nitrate-nitrite-nitric oxide pathway. Also, important from therapeutic point of view, all three organs are well responding to dietary nitrate interventions by increasing their respective nitrate levels, which leads to general increase of systemic and local NO and perhaps could be used as one of the ways to increase NO availability. Work supported by NIH internal grant to ANS. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diaphragm Force and Mitochondrial Function Following Enhanced Nitric Oxide Availability During Mechanical Ventilation

During mechanical ventilation (MV), force developed by the diaphragm decreases over time much faster than locomotor muscles, known as ventilator-induced diaphragm dysfunction (VIDD). VIDD is accompanied by an increase in intramyofiber protein S-nitrosylation, a modification of cysteines by nitric oxide (NO). During the treatment of acute respiratory distress syndrome (ARDS), inhaled NO is commonly used to improve gas exchange, but little is known about the effects of increased NO availability to the diaphragm during MV and the intracellular mechanisms to protect against excessive S-nitrosylation. We hypothesize that the enzyme S-nitrosoglutathione reductase (GSNOR) protects against excessive protein S-nitrosylation in diaphragm myofibers in conditions where NO availability is enhanced during MV, thereby preserving contractile function. We tested this hypothesis by enhancing NO availability and blocking GSNOR activity in mice during MV. Mice were treated with GSNOR inhibitor (SPL-334; GSNORi) or both GSNORi and NO donor isosorbide dinitrate (GSNORi-ISDN) together, and mechanically ventilated for 2h. E x vivo diaphragm force and mitochondrial respiration were measured. Male (C57BL/6J) 3-4 month old mice (n=27) were anesthetized and subjected to MV for 0 (non-MV), 2, 4, or 6h (150 breaths/min, 10 cmH2O PIP, 3.5 cmH2O PEEP. 8 mL/kg VT). Alternatively, mice were treated with PBS/10% DMSO (DMSO, n=6) or 25 μg SPL-334 in PBS/DMSO (GSNORi, n=6) or 25 μg SPL-334 + 1.7 mg ISDN in PBS/DMSO (GSNORi-ISDN; n=6), and then subjected to MV for 2 h. After MV, mice were euthanized, and diaphragm strips were used for force, and permeabilized fiber bundles for mitochondrial oxidative phosphorylation and H2O2 generation measurements. Peak tetanic force was decreased by MV ~23% at 4 h and ~40% at 6 h (P=0.0183 and P=0.0062, respectively, one-way ANOVA, Tukey post-test) vs 0 h. However, peak force was not different between DMSO, GSNORi and GSNORi-ISDN at 2 h MV. Coupled-phosphorylating mitochondrial respiration (Kruskal-Wallis H=2.2, p=0.35) was not different but H2O2 flux was highest in GSNORi-ISDN vs DMSO and GSNORi (Kruskal-Wallis H=6.6, p=0.03). GSNORi and ISDN treatment during MV did not produce any changes to VIDD and to mitochondrial respiration, but increased ROS production. If exogenous NO is not provided, inhibiting GSNOR in vivo alone does not affect diaphragm function ex vivo during short MV. Support: TRDRP (T32IR5221 and T29KT0397CA, to L.N.) and SDSU 2023 SEED Grant (to L.N.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reduction in blood pressure following acute dietary nitrate ingestion is correlated with increased red blood cell S-nitrosothiol concentrations

Dietary nitrate (NO3−) supplementation can enhance nitric oxide (NO) bioavailability and lower blood pressure (BP) in humans. The nitrite concentration ([NO2−]) in the plasma is the most commonly used biomarker of increased NO availability. However, it is unknown to what extent changes in other NO congeners, such as S-nitrosothiols (RSNOs), and in other blood components, such as red blood cells (RBC), also contribute to the BP lowering effects of dietary NO3−. We investigated the correlations between changes in NO biomarkers in different blood compartments and changes in BP variables following acute NO3− ingestion. Resting BP was measured and blood samples were collected at baseline, and at 1, 2, 3, 4 and 24 h following acute beetroot juice (∼12.8 mmol NO3−, ∼11 mg NO3−/kg) ingestion in 20 healthy volunteers. Spearman rank correlation coefficients were determined between the peak individual increases in NO biomarkers (NO3−, NO2−, RSNOs) in plasma, RBC and whole blood, and corresponding decreases in resting BP variables. No significant correlation was observed between increased plasma [NO2−] and reduced BP, but increased RBC [NO2−] was correlated with decreased systolic BP (rs = −0.50, P = 0.03). Notably, increased RBC [RSNOs] was significantly correlated with decreases in systolic (rs = −0.68, P = 0.001), diastolic (rs = −0.59, P = 0.008) and mean arterial pressure (rs = −0.64, P = 0.003). Fisher's z transformation indicated no difference in the strength of the correlations between increases in RBC [NO2−] or [RSNOs] and decreased systolic blood pressure. In conclusion, increased RBC [RSNOs] may be an important mediator of the reduction in resting BP observed following dietary NO3− supplementation.

Mitigation effect of galangin against aortic dysfunction and hypertrophy in rats with metabolic syndrome

Vascular alterations induced by a high-fat diet (HFD) are involved in the development of hypertension. Galangin, a flavonoid, is the major active compound isolated from galangal and propolis. The objective of this study was to investigate the effect of galangin on aortic endothelial dysfunction and hypertrophy, and the mechanisms involved in HFD-induced metabolic syndrome (MS) in rats. Male Sprague-Dawley rats (220–240 g) were separated into three groups: control + vehicle, MS + vehicle, and MS + galangin (50 mg/kg). Rats with MS received HFD plus 15% fructose solution for 16 weeks. Galangin or vehicle was orally administered daily for the final four weeks. Galangin reduced body weight and mean arterial pressure in HFD rats (p < 0.05). It also reduced circulating fasting blood glucose, insulin, and total cholesterol levels (p < 0.05). Impaired vascular responses to the exogenous acetylcholine observed in the aortic ring of HFD rats were restored by galangin (p < 0.05). However, the response to sodium nitroprusside did not differ between the groups. Galangin enhanced the expression of the aortic endothelial nitric oxide synthase (eNOS) protein and increased circulating nitric oxide (NO) levels in the MS group (p < 0.05). Aortic hypertrophy in HFD rats was alleviated by galangin (p < 0.05). Increases in tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6 levels, angiotensin-converting enzyme activity and angiotensin II (Ang II) concentrations in rats with MS were suppressed in galangin treated group (p < 0.05). Furthermore, galangin reduced the upregulation of angiotensin II type I receptor (AT1R) and transforming growth factor-beta (TGF-β) expression in rats with MS (p < 0.05). In conclusion, galangin alleviates metabolic disorders and improves aortic endothelial dysfunction and hypertrophy in the MS group. These effects were consistent with increased NO availability, reduced inflammation, and suppressing Ang II/AT1R/TGF-β signalling pathway.

Three main nitrate reservoirs in the mammalian body

Nitric oxide (NO) is a free radical with multiple functions in virtually all cells. In mammals, NO is generated by nitric oxide synthase from arginine and by reduction of nitrate/nitrite by tissue xanthine oxidoreductase (XOR) or microbiome. Previously, we reported in several mammalian species, including human, that skeletal muscle is a major nitrate reservoir in the body and that nitrate from this reservoir is systemically available and used to generate nitrite and NO. In our recent large animal study, we found that there are similar levels of nitrate stored in bone and skin. In our prior study, skeletal muscle showed a high level of nitrate at 57±39 nmol/g, relative to other tissues, such as liver and plasma at 15±5 nmol/g and 34±19 nmol/g, respectively. Here, we found that the average level of nitrate in the bone is 45±17 nmol/g and in the skin 41±19 nmol/g.Considering that the skeletal muscle, skin and skeleton are three largest organs in mammals, forming about 35-50%, 21% and 14% of body weight, respectively, these results highlight the importance of skeletal muscle as a main nitrate reservoir and the role of skin and bone as new auxiliary nitrate reservoirs. Along with skeletal muscle, the bones and skin likely play an important role in maintaining homeostasis of the nitrate-nitrite-nitric oxide pathway.Also, important from therapeutic point of view, all three organs are well responding to dietary nitrate interventions by increasing their respective nitrate levels, which leads to general increase of systemic and local NO availability and perhaps could be used as one of the ways to increase NO availability in some diseases. This work was supported by intramural NIDDK/NIH grant ZIA DK 025104-15 to Alan N. Schechter. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of Nitrates on Endurance Athletes- A Case Control Study

Beetroot itself is a precursor of Nitric Oxide (NO), it is used as a supplement. Increased NO availability will enhance oxygen and nutrient delivery to the working muscle by lowering the ATP cost of muscle contractile force production and the oxygen cost of aerobic exercise. The mechanisms contributed to these effects are linked to mitochondrial respiration and biogenesis which shows that enhancing NO bioavailability positively influences exercise performance in endurance athletes.VO2 max (maximal oxygen intake) is a familiar measurement related to aerobic endurance that numerous athletes use to determine their overall fitness as it measures the maximum amount of oxygen that a person can utilize throughout the intense exercise.

Plasma Nitrate Levels Are Related to Metabolic Syndrome and Are Not Altered by Treatment with DPP-4 Inhibitor Linagliptin: A Randomised, Placebo-Controlled Trial in Patients with Early Type 2 Diabetes Mellitus.

The depletion of nitrate and nitrite, stable nitric oxide (NO) end-products, promotes adipose tissue dysfunction and insulin resistance (IR). Dipeptidyl peptidase-4 (DPP-4) inhibitors have the potentially beneficial side effect of increasing NO availability. In this study, nitrate and nitrite levels and the effects of DPP-4 inhibitor linagliptin were investigated in relation to metabolic syndrome (MetS) markers. Treatment-naive patients with early type 2 diabetes mellitus (T2DM) (n = 40, median age 63 IQR (55–67) years, 63% male, mean HbA1c 45 ± 4.4 mmol/mol) were randomized (1:1) to linagliptin (5 mg/day) or placebo. MetS-related markers (body mass index (BMI), triglycerides, HOMA-IR, gamma-glutamyltransferase (GGT), C-reactive protein (CRP), and adiponectin), plasma levels of nitrate, nitrite, total free thiols (TFT) and vegetable intake were estimated at baseline and after 4 and 26 weeks of treatment. Plasma nitrate, but not nitrite, correlated positively with vegetable intake (r = 0.38, p = 0.018) and was inversely associated with HOMA-IR (r = −0.44, p = 0.006), BMI (r = −0.35, p = 0.028), GGT (r = −0.37, p = 0.019) and CRP (r = −0.34, p = 0.034). The relationship between nitrate and HOMA-IR remained significant after adjusting for BMI, CRP, vegetable intake and GGT. With stable vegetable intake, nitrate and nitrite, TFT, adipokines and CRP did not change after 26 weeks of linagliptin treatment. While plasma nitrate is inversely associated with MetS, linagliptin treatment does not significantly influence nitrate and nitrite concentrations, oxidative stress, adipose tissue function and systemic inflammation.

Acute supplementation with beetroot juice improves endothelial function in HIV-infected individuals.

Human immunodeficiency virus (HIV) is associated with lower nitric oxide (NO) bioavailability and vascular dysfunction. Nitrate-rich beetroot juice (BJ) has been shown to acutely increase NO availability and vascular function in healthy and individuals at high risk for cardiovascular disease. Thus, we tested the effects of BJ ingestion on flow-mediated dilation (FMD) and pulse wave velocity (PWV) measurements in healthy and HIV-infected patients. Thirteen HIV-infected individuals (age, 36 ± 10 years) and 18 healthy (age, 27 ± 8 years) participated in the study. Individuals were submitted to vascular tests such as FMD and pulse PWV at pre (T0) and at 120 min (T120) after BJ and placebo (PLA) ingestion. The %FMD at T0 of the control group was significantly higher than the %FMD at T0 of the HIV individuals in both interventions. BJ improved the %FMD at T120 when compared with T0 in the HIV and control groups. There was no change in %FMD after PLA ingestion in the control and HIV groups. There were no differences between groups (control vs HIV), time points (T0 vs T120), and interventions (BJ vs PLA) for PWV. Our findings showed that nitrate-rich BJ ingestion acutely improved vascular function in healthy and HIV-infected patients. Clinical Trials Registry no. NCT03485248. Novelty: HIV is associated with lower NO bioavailability and vascular dysfunction. Acute supplementation with nitrate-rich BJ has been shown to acutely increases NO bioavailability. We showed for the first time that BJ acutely improves endothelial function in HIV-infected patients.

Sickle cell disease subjects and mouse models have elevated nitrite and cGMP levels in blood compartments

The hypothesis of decreased nitric oxide (NO) bioavailability in sickle cell disease (SCD) proposes that multiple factors leading to decreased NO production and increased consumption contributes to vaso-occlusion, pulmonary hypertension, and pain. The anion nitrite is central to NO physiology as it is an end product of NO metabolism and serves as a reservoir for NO formation. However, there is little data on nitrite levels in SCD patients and its relationship to pain phenotype. We measured nitrite in SCD subjects and examined its relationship to SCD pain. In SCD subjects, median whole blood, red blood cell and plasma nitrite levels were higher than in controls, and were not associated with pain burden. Similarly, Townes and BERK homozygous SCD mice had elevated blood nitrite. Additionally, in red blood cells and plasma from SCD subjects and in blood and kidney from Townes homozygous mice, levels of cyclic guanosine monophosphate (cGMP) were higher compared to controls. In vitro, hemoglobin concentration, rather than sickle hemoglobin, was responsible for nitrite metabolism rate. In vivo, inhibition of NO synthases and xanthine oxidoreductase decreased nitrite levels in homozygotes but not in control mice. Long-term nitrite treatment in SCD mice further elevated blood nitrite and cGMP, worsened anemia, decreased platelets, and did not change pain response. These data suggest that SCD in humans and animals is associated with increased nitrite/NO availability, which is unrelated to pain phenotype. These findings might explain why multiple clinical trials aimed at increasing NO availability in SCD patients failed to improve pain outcomes.

Nitric oxide metabolites in hypoxia, freezing, and hibernation of the wood frog, Rana sylvatica.

Nitric oxide (NO) is a gaseous free radical that in diverse organisms performs many signaling and protective functions, such as vasoregulation, inhibition of apoptosis, antioxidation, and metabolic suppression. Increased availability of NO may be especially important during life-history periods when organisms contend with multiple stresses. We investigated dynamics of the NO metabolites, nitrite (NO2-) and nitrate (NO3-), in the blood plasma, heart, liver, and skeletal muscle of the wood frog (Rana sylvatica), an amphibian that endures chronic cold, freezing, hypoxia, dehydration, and extended aphagia during hibernation. We found elevated concentrations of NO2- and/or NO3- in the plasma (up to 4.1-fold), heart (3.1-fold), and liver (up to 4.1-fold) of frogs subjected to experimental hypoxia (24h, 4°C), and in the liver (up to 3.8-fold) of experimentally frozen frogs (48h, -2.5°C), suggesting that increased NO availability aids in survival of these stresses. During a 38-week period of simulated hibernation, NO2- and/or NO3- increased in the plasma (up to 10.4-fold), heart (up to 3.3-fold), and liver (5.0-fold) during an initial 5-week winter-acclimatization regimen and generally remained elevated thereafter. In hibernation, plasma NO2- was higher in frogs indigenous to Interior Alaska than in conspecifics from a temperate locale (southern Ohio), suggesting that NO availability is matched to the severity of environmental conditions prevailing in winter. The comparatively high NO availability in R. sylvatica, a stress-tolerant species, together with published values for other species, suggest that the NO protection system is of general importance in the stress adaptation of vertebrates.

The essential role of hypothalamic paraventricular nucleus nNOS in the modulation of autonomic control in exercised rats

Nitric oxide (NO), an intercellular signaling molecule is relevant for circulatory autonomic control. Brain NO synthase (NOS) and NO levels were downregulated in pathological conditions, but rescued after exercise training. We hypothesized that exercise training was also able to improve NO modulation within the hypothalamic paraventricular nucleus (PVN) of healthy rats. Male Wistar rats were submitted to two 4-weeks protocols: i) swimming training (T) or kept sedentary (S), ii) l-arginine (62,5 mg/mL, 1 mL/day p. o.) or vehicle supplementation. Rats underwent stereotaxic surgery (PVN bilateral guide cannulas) and chronic catheterization of artery/vein. Arterial pressure (AP), heart rate (HR) and baroreflex sensitivity were recorded in conscious rats at rest and following a selective nNOS inhibitor (Nw-Propyl-l-Arginine, 4 nmol/100 nL) within the PVN. Rats were deeply anesthetized for brain perfusion/harvesting after respiratory arrest. In separate groups (T and S, l-arginine and Vehicle supplemented) not submitted to PVN cannulation, fresh and fixed brains were obtained for gene and protein nNOS expression (qPCR and immunohistochemistry) and nitrite levels (Griess reaction). T and l-arginine treatment were accompanied by resting bradycardia, augmented parasympathetic and reduced sympathetic activity to heart and vessels (power spectral analysis) and increased baroreflex sensitivity (†P < 0.05). In contrast, PVN nNOS inhibition blocked/attenuated these effects in addition to significantly increase in resting MAP and HR (with larger effects in T and l-arginine treated rats vs. respective controls, †P < 0.05). T increased nNOS gene and protein expression within the ventromedial and posterior PVN nuclei (†P < 0.05). PVN nitirite levels were also increased in T and l-arginine groups (†P < 0.05). Data strongly suggest that training by increasing NO availability within PVN preautonomic nuclei favors both the slow down of sympathetic and the augmentation of parasympathetic activity and facilitates baroreflex control, therefore improving autonomic regulation of the heart in healthy rats.

Acute nitric oxide synthase inhibition enhances trans‐endothelial insulin efflux and muscle insulin sensitivity in vivo

Before insulin can stimulate muscle glucose uptake, it must first traverse the continuous endothelium of skeletal muscle (SkM) capillaries. Despite the importance of trans‐endothelial insulin transport to SkM insulin sensitivity, little is known about the regulation of this process in vivo. We previously developed a technique by which we can measure the rate of trans‐endothelial insulin efflux using intravital microscopy of a fully bioactive, fluorescent insulin probe (ins‐647). Using this technique, we showed that the movement of insulin across the endothelium in SkM occurs by a fluid‐phase transport mechanism which is not saturable and does not involve the insulin receptor. Furthermore, we have demonstrated that trans‐endothelial insulin efflux is impaired in obese, insulin resistant mice. Insulin resistance in humans and rodents is associated with endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability. Studies in cultured endothelial cells suggest that insulin transport by endothelial cells is positively regulated by NO. These findings led to the hypothesis that NO is a positive regulator of trans‐endothelial insulin efflux.To test this hypothesis, we treated male C57Bl/6 mice with an intravenous bolus of either 30 mg/kg L‐NG‐Nitroarginine methyl ester (L‐NAME; NO synthase inhibitor), or saline. L‐NAME treatment increased mean arterial blood pressure by ~30 mmHg, indicating that NO synthase was successfully inhibited. Intravital ins‐647 imaging demonstrated that L‐NAME enhanced the rate at which plasma insulin equilibrated with the interstitial space by ~25%, indicating enhanced trans‐endothelial insulin efflux. The more rapid trans‐endothelial movement of insulin was associated with a 25% increase in SkM insulin sensitivity as determined by the accumulation of 2[14C]2‐deoxyglucose (2DG) in the gastrocnemius. To test the effects of increasing NO availability on insulin efflux, mice were treated with 100 μg/kg/min sodium nitroprusside (SNP; NO donor) or saline. SNP had a small, but non‐significant blood pressure‐lowering effect. SNP had no effect on either trans‐endothelial insulin efflux kinetics or gastrocnemius 2DG accumulation. To test whether the effects of NO modulation were due to changes in blood pressure, we infused a primed, continuous infusion of either phenylephrine (300 μg/kg bolus; 300 μg/kg/min infusion) or hydralazine (1 mg/kg bolus; 1 mg/kg/min infusion) into mice. Phenylephrine and hydralazine are direct smooth‐muscle acting drugs which raise and lower blood pressure independent of NO, respectively. Intravital ins‐647 imaging demonstrated that neither phenylephrine nor hydralazine had any effect on trans‐endothelial insulin efflux. Taken together, these results suggest that, surprisingly, reduction of NO by L‐NAME treatment increases trans‐endothelial insulin efflux. The mechanism by which lowering NO enhances the movement of insulin may involve effects of L‐NAME on SkM hemodynamics or direct effects on the capillary endothelium.Support or Funding InformationThis work was supported by NIH grants R01DK054902 and U24 DK059637 to D.H.W.; grant F31DK109594 to I.M.W.; and grant T32DK007563 to I.M.W.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Muscle synergies and activation in Nordic walking compared with conventional walking

Nitric oxide (NO), an intercellular signaling molecule is relevant for circulatory autonomic control. Brain NO synthase (NOS) and NO levels were downregulated in pathological conditions, but rescued after exercise training. We hypothesized that exercise training was also able to improve NO modulation within the hypothalamic paraventricular nucleus (PVN) of healthy rats. Male Wistar rats were submitted to two 4-weeks protocols: i) swimming training (T) or kept sedentary (S), ii) l-arginine (62,5 mg/mL, 1 mL/day p. o.) or vehicle supplementation. Rats underwent stereotaxic surgery (PVN bilateral guide cannulas) and chronic catheterization of artery/vein. Arterial pressure (AP), heart rate (HR) and baroreflex sensitivity were recorded in conscious rats at rest and following a selective nNOS inhibitor (Nw-Propyl-l-Arginine, 4 nmol/100 nL) within the PVN. Rats were deeply anesthetized for brain perfusion/harvesting after respiratory arrest. In separate groups (T and S, l-arginine and Vehicle supplemented) not submitted to PVN cannulation, fresh and fixed brains were obtained for gene and protein nNOS expression (qPCR and immunohistochemistry) and nitrite levels (Griess reaction). T and l-arginine treatment were accompanied by resting bradycardia, augmented parasympathetic and reduced sympathetic activity to heart and vessels (power spectral analysis) and increased baroreflex sensitivity (†P < 0.05). In contrast, PVN nNOS inhibition blocked/attenuated these effects in addition to significantly increase in resting MAP and HR (with larger effects in T and l-arginine treated rats vs. respective controls, †P < 0.05). T increased nNOS gene and protein expression within the ventromedial and posterior PVN nuclei (†P < 0.05). PVN nitirite levels were also increased in T and l-arginine groups (†P < 0.05). Data strongly suggest that training by increasing NO availability within PVN preautonomic nuclei favors both the slow down of sympathetic and the augmentation of parasympathetic activity and facilitates baroreflex control, therefore improving autonomic regulation of the heart in healthy rats.

Effect of acute nitrate and citrulline supplementation on muscle microvascular response to ischemia-reperfusion in healthy humans.

Nitric oxide (NO) is implicated in vasomotor control mechanisms altering the diameter of the vessels under various physiological and pathological conditions. There are 2 main NO production pathways, 1 NO synthase (NOS) independent (nitrate-nitrite-NO) and the other is NOS dependent (citrulline-arginine-NO). The objective of the study was to evaluate the effect of acute nitrate and citrulline supplementation on post-ischemic vascular response in healthy subjects. Fourteen subjects performed 2-leg vascular occlusion tests, 3 days apart. They were randomly assigned to consume a drink containing 1200 mg (19.4 mmol) of nitrate and 6 g of citrulline (N+C) or a placebo (Pl). Changes in total hemoglobin (Hbtot) and oxyhemoglobin (HbO2) concentrations were recorded by near-infrared spectroscopy on the thigh and calf muscles. No differences between N+C and Pl were observed during the ischemic period. Hbtot increased to a larger extent during the reperfusion period for the thigh (e.g., area under the curve, 821 ± 324 vs. 627 ± 381 mmol·s-1, p = 0.003) and the calf (515 ± 285 vs. 400 ± 275 mmol·s-1, p = 0.029) in the N+C versus Pl conditions. Similar results were found regarding HbO2 for the thigh (e.g., area under the curve, 842 ± 502 vs. 770 ± 491 mmol·s-1, p = 0.077) and the calf (968 ± 536 vs. 865 ± 275 mmol·s-1, p = 0.075). The larger postocclusive Hbtot and HbO2 responses observed after N+C intake suggests a greater post-ischemic vasodilation, which may be due to increased NO availability, via the activation of the 2 main NO production pathways.

Nitrite Infusion Does Not Alter Isolated Canine Muscle Oxidative Metabolism In Situ During Hypoxia With Normal Convective O2 Delivery

IntroductionPrevious studies have shown a decreased muscle oxygen consumption (V′O2) during moderate intensity exercise in hypoxia after augmented nitric oxide (NO) bioavailability, which suggests an increased efficiency of oxidative metabolism. It is not clear if these effects are related to an NO‐induced increase in convective O2 delivery and/or to an enhanced mitochondrial coupling efficiency. The aim of this study was to examine skeletal muscle contraction economy during hypoxic exercise following nitrite infusion with unchanged convective O2 delivery.MethodsCanine gastrocnemius muscles (n=8) were surgically isolated and electrically stimulated via the sciatic nerve, eliciting 1 contraction every 3 seconds (≈35% V′O2peak) for 3 minutes in hypoxic conditions (PaO2=42±3 mmHg). During contractions, sodium nitrite (NITR) or sodium chloride (SAL) was infused into the popliteal artery. Muscle blood flow was kept constant by a perfusion pump. Muscle force was measured and muscle V′O2 was calculated from the Fick principle. Muscle biopsies were obtained before and after NITR and were used to isolate mitochondria. Mitochondrial respiration rates were recorded by high‐resolution respirometry.ResultsMuscle V′O2 was not significantly different between NITR (60.6±18.0 ml/kg/min) and SAL (61.7±18.3ml/kg/min). After normalizing V′O2 per unit of developed force, the values were slightly but not significantly higher in NITR vs. SAL (p=0.10). No differences were found for ADP‐stimulated mitochondrial respiration (both for complex I and complex II), leak respiration and oxidative phosphorylation coupling.ConclusionIn hypoxic conditions, but in the presence of constant and normal convective O2 delivery, nitrite infusion did not affect canine skeletal muscle oxidative metabolism. These results suggest that the effects of increased NO availability on muscle contraction efficiency in hypoxia, if present, are likely not attributable to changes in mitochondrial respiratory function.

Dose-dependent Effects Of Uv-a Light On Nitric Oxide Bioavailability And Cardiometabolic Function In Humans

Exposing human skin to 20 J·cm2 of ultraviolet-A (UV-A) light has been shown to release nitric oxide (NO) species from dermal storage forms and mildly reduce blood pressure (BP). The dose-response relationship of these effects, however, remains unknown.PURPOSE: To determine the effects of two different doses of UV-A light on NO metabolites, mean arterial pressure (MAP) and resting energy expenditure (REE).METHODS: Eight healthy males (age: 28 ± 5 yr, body mass: 81 ± 12 kg) were exposed to three light conditions in a randomised counter-balanced order. Following at least 30 min of supine rest, participants were exposed to either (i) no light (CON) (ii) 10 J·cm2 UV-A light (UVA10), or (iii) 20 J·cm2 UV-A light (UVA20). UVA20 is comparable to 30 min of sunshine in southern Europe in summer. During the experiment oxygen consumption (VO2) and REE were monitored continuously using indirect calorimetry. Regular BP measurements were taken and samples of venous blood collected before and after light exposure. Plasma nitrate [NO3-] and nitrite [NO2-] were later analysed via HPLC (n=7). Statistical differences are reported together with effect sizes (Cohen?s d) where appropriate.RESULTS: There were no significant changes in plasma [NO3-] or [NO2-] between pre- and post-exposures in all conditions (P>0.05). Plasma [NO2-] was moderately higher after UVA20 exposure compared to CON (? 29%, 178 ± 299 nM, d=0.58, P=0.17) but increased to only a small extent with UV10 (? 9%, 58 ± 220 nM, d=0.26, P=0.51). There were no differences in MAP between conditions or before and after exposure (both P>0.50). There was a marked decline in REE following UVA20 (? 7%, 112 ± 143 kcal/day, d=0.78, P<0.01) and UV10 (? 4% 134± 159 kcal/day, d=0.84, P=0.07) while no significant differences were observed in the CON group (P=1.0). From pre- to post-light exposure, VO2 declined with UVA20 (? 6%, 13 ± 19 ml/min, P=0.04), but did not change with UVA10 (P=0.26) or CON (P=0.44).CONCLUSION: The present data suggests that a 20 J·cm2 dose of UV-A light increases [NO2-] and decreases REE and VO2 to a greater extent than 10 J·cm2. Although speculative, it is probable that reductions in REE and VO2 were mediated by an increased NO availability. Neither dose of UV-A light resulted in a detectable change in BP although a larger sample is required before definitive conclusions can be made.

Tissue inflammation and nitric oxide-mediated alterations in cardiovascular function are major determinants of endotoxin-induced insulin resistance.

BackgroundEndotoxin (i.e. LPS) administration induces a robust inflammatory response with accompanying cardiovascular dysfunction and insulin resistance. Overabundance of nitric oxide (NO) contributes to the vascular dysfunction. However, inflammation itself also induces insulin resistance in skeletal muscle. We sought to investigate whether the cardiovascular dysfunction induced by increased NO availability without inflammatory stress can promote insulin resistance. Additionally, we examined the role of inducible nitric oxide synthase (iNOS or NOS2), the source of the increase in NO availability, in modulating LPS-induced decrease in insulin-stimulated muscle glucose uptake (MGU).MethodsThe impact of NO donor infusion on insulin-stimulated whole-body and muscle glucose uptake (hyperinsulinemic-euglycemic clamps), and the cardiovascular system was assessed in chronically catheterized, conscious mice wild-type (WT) mice. The impact of LPS on insulin action and the cardiovascular system were assessed in WT and global iNOS knockout (KO) mice. Tissue blood flow and cardiac function were assessed using microspheres and echocardiography, respectively. Insulin signaling activity, and gene expression of pro-inflammatory markers were also measured.ResultsNO donor infusion decreased mean arterial blood pressure, whole-body glucose requirements, and MGU in the absence of changes in skeletal muscle blood flow. LPS lowered mean arterial blood pressure and glucose requirements in WT mice, but not in iNOS KO mice. Lastly, despite an intact inflammatory response, iNOS KO mice were protected from LPS-mediated deficits in cardiac output. LPS impaired MGU in vivo, regardless of the presence of iNOS. However, ex vivo, insulin action in muscle obtained from LPS treated iNOS KO animals was protected.ConclusionNitric oxide excess and LPS impairs glycemic control by diminishing MGU. LPS impairs MGU by both the direct effect of inflammation on the myocyte, as well as by the indirect NO-driven cardiovascular dysfunction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12933-015-0223-2) contains supplementary material, which is available to authorized users.

NO Better Way to Protect the Heart during Ischemia-Reperfusion: To be in the Right Place at the Right Time.

Acute myocardial infarction (MI) is one of the leading causes of mortality worldwide. MI is the heart muscle irreversible death secondary to prolonged ischemia. Over the last few decades, medical progress in how and when to restore blood flow to the ischemic area have markedly improved patient survival. Although early heart reperfusion is acknowledged to be the most effective way to limit infarct size, post-ischemic reperfusion is associated with detrimental effects, such as myocardial stunning, ventricular arrhythmias, microvascular dysfunction, and cell death. The molecular mechanisms of these reperfusion injuries remain to be elucidated and their management is very challenging. Among the various therapeutic molecular approaches proposed by experimental studies, nitric oxide (NO) role in protecting heart against MI and reperfusion injuries has been widely assessed and discussed (1–4). NO is a gasotransmitter that is abundantly produced in the cardiovascular system mainly by the NO synthase (NOS) enzymes system. Two isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS), are constitutively expressed in both myocardium and vessels, whereas inducible NOS (iNOS) is detected only in pathological conditions, such as inflammatory and/or oxidative stress. Both eNOS and nNOS are low-NO output Ca2+-dependent enzymes, while iNOS is a high-NO output Ca2+-independent enzyme. In physiological conditions, NOS form homodimers (“coupled” NOS) that catalyze NO production from l-arginine and O2 through electron transfer from NADPH on the reductase domain of one monomer to the oxidase domain of the second monomer. In pathological conditions, such as in the absence of the essential cofactor tetrahydrobiopterin (BH4), eNOS can be “uncoupled” to produce O2− instead of NO. In stress conditions, NO protects tissues through two distinct pathways. In the first one, NO activates the soluble guanylate cyclase (sGC) that initiates cyclic guanosine monophosphate (cGMP) production, leading to the activation of protein kinase G (PKG). As sGC is the major cell receptor for NO and the NO/sGC/cGMP/PKG pathway plays a critical role in both myocardium excitation–contraction coupling and cardiovascular function regulation (5–8), NO cardioprotective role was first attributed to PKG activation (9–11). However, a second pathway in which proteins are directly modified by NO addition to sulfhydryl residues, a process known as S-nitrosylation (SNO), has recently emerged in the scientific literature. Although PKG activation pathway has been largely involved in NO-mediated cardioprotection (11–13), SNO is now taking the front stage and is considered to be a key player in cardioprotection through (i) the transient modification of protein activity and/or (ii) their protection from irreversible oxidation (14–17). Indeed, Sun et al. (18) showed that reduced heart vulnerability to ischemia–reperfusion (IR) following acute ischemic preconditioning is mainly related to SNO signaling and not to PKG activation through the NO–SGC–cGMP pathway. Accordingly, we found that in exercise training-induced cardioprotection against IR injuries, protein SNO level, but not cGMP level, increased during early reperfusion (19). The same year, Methner et al. (20), using a Cre/loxP approach to selectively ablate type I PKG in cardiomyocytes, demonstrated that ischemic post-conditioning reduced infarct size in these mice like in wild type controls. Moreover, they showed that the cardioprotective effect against IR injury of mitochondria-targeted S-nitrosothiol (MitoSNO), which allows NO and S-nitrosothiol accumulation in mitochondria, was comparable in mice that specifically lack PKG in cardiomyocytes and in controls. This indicates that MitoSNO cardioprotective effect is independent of PKG. The current literature strongly supports NO implication in cardioprotection. However, the mechanism is still debated and whether increased NO availability during IR is cytoprotective remains to be demonstrated. Here, we discuss how NO might contribute to protect heart and particularly the importance of NO (i) localization, (ii) concentration, and (iii) time of availability during IR.

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO–RBCs interactions in ‘competition experiments’. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO–RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

VO2 Kinetics in Supra-Anaerobic Threshold Constant Tests Allow the Visualization and Quantification of the O2 Saving After Cytochrome C Oxidase Inhibition by Aerobic Training or Nitrate Administration

We tested whether the known cytochrome c oxidase (COX) inhibition by nitric oxide (NO) could be quantified by VO(2) kinetics during constant load supra-Anaerobic Threshold (AT) exercises in healthy trained or untrained subjects following aerobic training or nitrate administration. In cycle ergometer constant load exercises supra-AT, identified in previous incremental tests, VO(2) kinetics describe a double exponential curve, one rapid and one appreciably slower, allowing the area between them to be calculate in O(2) l. After training, with increased NO availability, this area decreases in inverse ratio to treatment efficacy. In fact, in 11 healthy subjects after aerobic training for 6-7 weeks, area was decreased on average by 51 %. In 11 untrained subjects, following the assumption of an NO donor, 20 mg isosorbide 5 mononitrate, area was decreased on average by 53 %. In conclusion, supra-AT VO(2) kinetics in constant load exercises permit the quantification of the inhibitory effect NO-dependent on COX after either physical training or nitrate assumption.