Dietary Nitrate Metabolism in Porcine Ocular Tissues Determined Using 15N-Labeled Sodium Nitrate Supplementation.

Nitrate (NO3-) obtained from the diet is converted to nitrite (NO2-) and subsequently to nitric oxide (NO) within the body. Previously, we showed that porcine eye components contain substantial amounts of nitrate and nitrite that are similar to those in blood. Notably, cornea and sclera exhibited the capability to reduce nitrate to nitrite. To gain deeper insights into nitrate metabolism in porcine eyes, our current study involved feeding pigs either NaCl or Na15NO3 and assessing the levels of total and 15N-labeled NO3-/NO2- in various ocular tissues. Three hours after Na15NO3 ingestion, a marked increase in 15NO3- and 15NO2- was observed in all parts of the eye; in particular, the aqueous and vitreous humor showed a high 15NO3- enrichment (77.5 and 74.5%, respectively), similar to that of plasma (77.1%) and showed an even higher 15NO2- enrichment (39.9 and 35.3%, respectively) than that of plasma (19.8%). The total amounts of NO3- and NO2- exhibited patterns consistent with those observed in 15N analysis. Next, to investigate whether nitrate or nitrite accumulate proportionally after multiple nitrate treatments, we measured nitrate and nitrite contents after supplementing pigs with Na15NO3 for five consecutive days. In both 15N-labeled and total nitrate and nitrite analysis, we did not observe further accumulation of these ions after multiple treatments, compared to a single treatment. These findings suggest that dietary nitrate supplementation exerts a significant influence on nitrate and nitrite levels and potentially NO levels in the eye and opens up the possibility for the therapeutic use of dietary nitrate/nitrite to enhance or restore NO levels in ocular tissues.

Nitrate and Nitrite Metabolism in Aging Rats: A Comparative Study.

Nitric oxide (NO) (co)regulates many physiological processes in the body. Its short-lived free radicals force synthesis in situ and on-demand, without storage possibility. Local oxygen availability determines the origin of NO-either by synthesis by nitric oxide synthases (NOS) or by the reduction of nitrate to nitrite to NO by nitrate/nitrite reductases. The existence of nitrate reservoirs, mainly in skeletal muscle, assures the local and systemic availability of NO. Aging is accompanied by changes in metabolic pathways, leading to a decrease in NO availability. We explored age-related changes in various rat organs and tissues. We found differences in nitrate and nitrite contents in tissues of old and young rats at baseline levels, with nitrate levels being generally higher and nitrite levels being generally lower in old rats. However, there were no differences in the levels of nitrate-transporting proteins and nitrate reductase between old and young rats, with the exception of in the eye. Increased dietary nitrate led to significantly higher nitrate enrichment in the majority of old rat organs compared to young rats, suggesting that the nitrate reduction pathway is not affected by aging. We hypothesize that age-related NO accessibility changes originate either from the NOS pathway or from changes in NO downstream signaling (sGC/PDE5). Both possibilities need further investigation.

Distribution of dietary nitrate and its metabolites in rat tissues after 15N-labeled nitrate administration.

The reduction pathway of nitrate (NO3-) and nitrite (NO2-) to nitric oxide (NO) contributes to regulating many physiological processes. To examine the rate and extent of dietary nitrate absorption and its reduction to nitrite, we supplemented rat diets with Na15NO3-containing water (1 g/L) and collected plasma, urine and several tissue samples. We found that plasma and urine showed 8.8- and 11.7-fold increases respectively in total nitrate concentrations in 1-day supplementation group compared to control. In tissue samples-gluteus, liver and eyes-we found 1.7-, 2.4- and 4.2-fold increases respectively in 1-day supplementation group. These increases remained similar in 3-day supplementation group. LC-MS/MS analysis showed that the augmented nitrate concentrations were primarily from the exogenously provided 15N-nitrate. Overall nitrite concentrations and percent of 15N-nitrite were also greatly increased in all samples after nitrate supplementation; eye homogenates showed larger increases compared to gluteus and liver. Moreover, genes related to nitrate transport and reduction (Sialin, CLC and XOR) were upregulated after nitrate supplementation for 3 days in muscle (Sialin 2.3-, CLC1 1.3-, CLC3 2.1-, XOR 2.4-fold) and eye (XOR 1.7-fold) homogenates. These results demonstrate that dietary nitrate is quickly absorbed into circulation and tissues, and it can be reduced to nitrite in tissues (and likely to NO) suggesting that nitrate-enriched diets can be an efficient intervention to enhance nitrite and NO bioavailability.

15 N-labeled dietary nitrate supplementation increases human skeletal muscle nitrate concentration and improves muscle torque production.

AIM: Dietary nitrate (NO3 - ) supplementation increases nitric oxide bioavailability and can enhance exercise performance. We investigated the distribution and metabolic fate of ingested NO3 - at rest and during exercise with a focus on skeletal muscle. METHODS: In a randomized, crossover study, 10 healthy volunteers consumed 12.8 mmol 15 N-labeled potassium nitrate (K15 NO3 ; NIT) or potassium chloride placebo (PLA). Muscle biopsies were taken at baseline, at 1- and 3-h post-supplement ingestion, and immediately following the completion of 60 maximal intermittent contractions of the knee extensors. Muscle, plasma, saliva, and urine samples were analyzed using chemiluminescence to determine absolute [NO3 - ] and [NO2 - ], and by mass spectrometry to determine the proportion of NO3 - and NO2 - that was 15 N-labeled. RESULTS: Neither muscle [NO3 - ] nor [NO2 - ] were altered by PLA. Following NIT, muscle [NO3 - ] (but not [NO2 - ]) was elevated at 1-h (from ~35 to 147 nmol/g, p < 0.001) and 3-h, with almost all of the increase being 15 N-labeled. There was a significant reduction in 15 N-labeled muscle [NO3 - ] from pre- to post-exercise. Relative to PLA, mean muscle torque production was ~7% greater during the first 18 contractions following NIT. This improvement in torque was correlated with the pre-exercise 15 N-labeled muscle [NO3 - ] and the magnitude of decline in 15 N-labeled muscle [NO3 - ] during exercise (r = 0.66 and r = 0.62, respectively; p < 0.01). CONCLUSION: This study shows, for the first time, that skeletal muscle rapidly takes up dietary NO3 - , the elevated muscle [NO3 - ] following NO3 - ingestion declines during exercise, and muscle NO3 - dynamics are associated with enhanced torque production during maximal intermittent muscle contractions.

Human skeletal muscle nitrate and nitrite in individuals with peripheral arterial disease: Effect of inorganic nitrate supplementation and exercise.

Skeletal muscle may act as a reservoir for N-oxides following inorganic nitrate supplementation. This idea is most intriguing in individuals with peripheral artery disease (PAD) who are unable to endogenously upregulate nitric oxide. This study analyzed plasma and skeletal muscle nitrate and nitrite concentrations along with exercise performance, prior to and following 12-weeks of exercise training combined with oral inorganic nitrate supplementation (EX+BR) or placebo (EX+PL) in participants with PAD. Non-supplemented, at baseline, there were no differences in plasma and muscle nitrate. For nitrite, muscle concentration was higher than plasma (+0.10 nmol.g-1 ). After 12 -weeks, acute oral nitrate increased both plasma and muscle nitrate (455.04 and 121.14 nmol.g-1 , p < 0.01), which were correlated (r = 0.63, p < 0.01), plasma nitrate increase was greater than in muscle (p < 0.01). Nitrite increased in the plasma (1.01 nmol.g-1 , p < 0.05) but not in the muscle (0.22 nmol.g-1 ) (p < 0.05 between compartments). Peak walk time (PWT) increased in both groups (PL + 257.6 s;BR + 315.0 s). Six-minute walk (6 MW) distance increased only in the (EX+BR) group (BR + 75.4 m). We report no substantial gradient of nitrate (or nitrite) from skeletal muscle to plasma, suggesting a lack of reservoir-like function in participants with PAD. Oral nitrate supplementation produced increases in skeletal muscle nitrate, but not skeletal muscle nitrite. The related changes in nitrate concentration between plasma and muscle suggests a potential for inter-compartmental nitrate "communication". Skeletal muscle did not appear to play a role in within compartment nitrate reduction. Muscle nitrate and nitrite concentrations did not appear to contribute to exercise performance in patients with PAD.

Dietary Inorganic Nitrate as an Ergogenic Aid: An Expert Consensus Derived via the Modified Delphi Technique.

INTRODUCTION: Dietary inorganic nitrate is a popular nutritional supplement, which increases nitric oxide bioavailability and may improve exercise performance. Despite over a decade of research into the effects of dietary nitrate supplementation during exercise there is currently no expert consensus on how, when and for whom this compound could be recommended as an ergogenic aid. Moreover, there is no consensus on the safe administration of dietary nitrate as an ergogenic aid. This study aimed to address these research gaps. METHODS: The modified Delphi technique was used to establish the views of 12 expert panel members on the use of dietary nitrate as an ergogenic aid. Over three iterative rounds (two via questionnaire and one via videoconferencing), the expert panel members voted on 222 statements relating to dietary nitrate as an ergogenic aid. Consensus was reached when > 80% of the panel provided the same answer (i.e. yes or no). Statements for which > 80% of the panel cast a vote of insufficient evidence were categorised as such and removed from further voting. These statements were subsequently used to identify directions for future research. RESULTS: The 12 panel members contributed to voting in all three rounds. A total of 39 statements (17.6%) reached consensus across the three rounds (20 yes, 19 no). In round one, 21 statements reached consensus (11 yes, 10 no). In round two, seven further statements reached consensus (4 yes, 3 no). In round three, an additional 11 statements reached consensus (5 yes, 6 no). The panel agreed that there was insufficient evidence for 134 (60.4%) of the statements, and were unable to agree on the outcome of the remaining statements. CONCLUSIONS: This study provides information on the current expert consensus on dietary nitrate, which may be of value to athletes, coaches, practitioners and researchers. The effects of dietary nitrate appear to be diminished in individuals with a higher aerobic fitness (peak oxygen consumption [V̇O2peak] > 60 ml/kg/min), and therefore, aerobic fitness should be taken into account when considering use of dietary nitrate as an ergogenic aid. It is recommended that athletes looking to benefit from dietary nitrate supplementation should consume 8-16 mmol nitrate acutely or 4-16 mmol/day nitrate chronically (with the final dose ingested 2-4 h pre-exercise) to maximise ergogenic effects, taking into consideration that, from a safety perspective, athletes may be best advised to increase their intake of nitrate via vegetables and vegetable juices. Acute nitrate supplementation up to ~ 16 mmol is believed to be safe, although the safety of chronic nitrate supplementation requires further investigation. The expert panel agreed that there was insufficient evidence for most of the appraised statements, highlighting the need for future research in this area.

Time course of human skeletal muscle nitrate and nitrite concentration changes following dietary nitrate ingestion.

Dietary nitrate (NO3-) ingestion can be beneficial for health and exercise performance. Recently, based on animal and limited human studies, a skeletal muscle NO3- reservoir has been suggested to be important in whole body nitric oxide (NO) homeostasis. The purpose of this study was to determine the time course of changes in human skeletal muscle NO3- concentration ([NO3-]) following the ingestion of dietary NO3-. Sixteen participants were allocated to either an experimental group (NIT: n = 11) which consumed a bolus of ∼1300 mg (12.8 mmol) potassium nitrate (KNO3), or a placebo group (PLA: n = 5) which consumed a bolus of potassium chloride (KCl). Biological samples (muscle (vastus lateralis), blood, saliva and urine) were collected shortly before NIT or PLA ingestion and at intervals over the course of the subsequent 24 h. At baseline, no differences were observed for muscle [NO3-] and [NO2-] between NIT and PLA (P > 0.05). In PLA, there were no changes in muscle [NO3-] or [NO2-] over time. In NIT, muscle [NO3-] was significantly elevated above baseline (54 ± 29 nmol/g) at 0.5 h, reached a peak at 3 h (181 ± 128 nmol/g), and was not different to baseline from 9 h onwards (P > 0.05). Muscle [NO2-] did not change significantly over time. Following ingestion of a bolus of dietary NO3-, skeletal muscle [NO3-] increases rapidly, reaches a peak at ∼3 h and subsequently declines towards baseline values. Following dietary NO3- ingestion, human m. vastus lateralis [NO3-] expressed a slightly delayed pharmacokinetic profile compared to plasma [NO3-].

Skeletal Muscle Nitrate as a Regulator of Systemic Nitric Oxide Homeostasis.

Nonenzymatic nitric oxide (NO) generation via the reduction of nitrate and nitrite ions, along with remarkably high levels of nitrate ions in skeletal muscle, have been described recently. Skeletal muscle nitrate storage may be critical for maintenance of NO homeostasis in healthy aging, and nitrate supplementation may be useful for the treatment of specific pathophysiologies and for enhancing normal functions.

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

Control of rat muscle nitrate levels after perturbation of steady state dietary nitrate intake.

The roles of nitrate and nitrite ions as nitric oxide (NO) sources in mammals, complementing NOS enzymes, have recently been the focus of much research. We previously reported that rat skeletal muscle serves as a nitrate reservoir, with the amount of stored nitrate being highly dependent on dietary nitrate availability, as well as its synthesis by NOS1 enzymes and its subsequent utilization. We showed that at conditions of increased NO need, this nitrate reservoir is used in situ to generate nitrite and NO, at least in part via the nitrate reductase activity of xanthine oxidoreductase (XOR). We now further investigate the dynamics of nitrate/nitrite fluxes in rat skeletal muscle after first increasing nitrate levels in drinking water and then returning to the original intake level. Nitrate/nitrite levels were analyzed in liver, blood and several skeletal muscle samples, and expression of proteins involved in nitrate metabolism and transport were also measured. Increased nitrate supply elevated nitrate and nitrite levels in all measured tissues. Surprisingly, after high nitrate diet termination, levels of both ions in liver and all muscle samples first declined to lower levels than the original baseline. During the course of the overall experiment there was a gradual increase of XOR expression in muscle tissue, which likely led to enhanced nitrate to nitrite reduction. We also noted differences in basal levels of nitrate in the different types of muscles. These findings suggest complex control of muscle nitrate levels, perhaps with multiple processes to preserve its intracellular levels.

Dietary Nitrate and Nitric Oxide Metabolism: Mouth, Circulation, Skeletal Muscle, and Exercise Performance.

Nitric oxide (NO) is a gaseous signaling molecule that plays an important role in myriad physiological processes, including the regulation of vascular tone, neurotransmission, mitochondrial respiration, and skeletal muscle contractile function. NO may be produced via the canonical NO synthase-catalyzed oxidation of l-arginine and also by the sequential reduction of nitrate to nitrite and then NO. The body's nitrate stores can be augmented by the ingestion of nitrate-rich foods (primarily green leafy vegetables). NO bioavailability is greatly enhanced by the activity of bacteria residing in the mouth, which reduce nitrate to nitrite, thereby increasing the concentration of circulating nitrite, which can be reduced further to NO in regions of low oxygen availability. Recent investigations have focused on promoting this nitrate-nitrite-NO pathway to positively affect indices of cardiovascular health and exercise tolerance. It has been reported that dietary nitrate supplementation with beetroot juice lowers blood pressure in hypertensive patients, and sodium nitrite supplementation improves vascular endothelial function and reduces the stiffening of large elastic arteries in older humans. Nitrate supplementation has also been shown to enhance skeletal muscle function and to improve exercise performance in some circumstances. Recently, it has been established that nitrate concentration in skeletal muscle is much higher than that in blood and that muscle nitrate stores are exquisitely sensitive to dietary nitrate supplementation and deprivation. In this review, we consider the possibility that nitrate represents an essential storage form of NO and discuss the integrated function of the oral microbiome, circulation, and skeletal muscle in nitrate-nitrite-NO metabolism, as well as the practical relevance for health and performance.

Potential roles of nitrate and nitrite in nitric oxide metabolism in the eye.

Nitric oxide (NO) signaling has been studied in the eye, including in the pathophysiology of some eye diseases. While NO production by nitric oxide synthase (NOS) enzymes in the eye has been characterized, the more recently described pathways of NO generation by nitrate (NO3-) and nitrite (NO2-) ions reduction has received much less attention. To elucidate the potential roles of these pathways, we analyzed nitrate and nitrite levels in components of the eye and lacrimal glands, primarily in porcine samples. Nitrate and nitrite levels were higher in cornea than in other eye parts, while lens contained the least amounts. Lacrimal glands exhibited much higher levels of both ions compared to other organs, such as liver and skeletal muscle, and even to salivary glands which are known to concentrate these ions. Western blotting showed expression of sialin, a known nitrate transporter, in the lacrimal glands and other eye components, and also xanthine oxidoreductase, a nitrate and nitrite reductase, in cornea and sclera. Cornea and sclera homogenates possessed a measurable amount of nitrate reduction activity. These results suggest that nitrate ions are concentrated in the lacrimal glands by sialin and can be secreted into eye components via tears and then reduced to nitrite and NO, thereby being an important source of NO in the eye.

Nitrate uptake and metabolism in human skeletal muscle cell cultures.

Several studies show that dietary nitrate enhances exercise performance, presumably by increasing muscle blood flow and improving oxygen utilization. These effects are likely mediated by nitrate metabolites, including nitrite and nitric oxide (NO). However, the mechanisms of nitrate production, storage, and metabolism to nitrite and NO in skeletal muscle cells are still unclear. We hypothesized that exogenous nitrate can be taken up and metabolized to nitrite/NO inside the skeletal muscle. We found rapid uptake of exogeneous nitrate in both myoblasts and myotubes, increasing nitrite levels in myotubes, but not myoblasts. During differentiation we found increased expression of molybdenum containing proteins, such as xanthine oxidoreductase (XOR) and the mitochondrial amidoxime-reducing component (MARC); nitrate and nitrite reductases. Sialin, a known nitrate transporter, was detected in myoblasts; nitrate uptake decreased after sialin knockdown. Inhibition of chloride channel 1 (CLC1) also led to significantly decreased uptake of nitrate. Addition of exogenous nitrite, which resulted in higher intracellular nitrite levels, increased intracellular cGMP levels in myotubes. In summary, our results demonstrate for the first time the presence of the nitrate/nitrite/NO pathway in skeletal muscle cells, namely the existence of strong uptake of exogenous nitrate into cells and conversion of intracellular nitrate to nitrite and NO. Our results further support our previously formulated hypothesis about the importance of the nitrate to nitrite to NO intrinsic reduction pathways in skeletal muscles, which likely contributes to improved exercise tolerance after nitrate ingestion.

Human skeletal muscle nitrate store: influence of dietary nitrate supplementation and exercise.

KEY POINTS: Nitric oxide (NO), a potent vasodilator and a regulator of many physiological processes, is produced in mammals both enzymatically and by reduction of nitrite and nitrate ions. We have previously reported that, in rodents, skeletal muscle serves as a nitrate reservoir, with nitrate levels greatly exceeding those in blood or other internal organs, and with nitrate being reduced to NO during exercise. In the current study, we show that nitrate concentration is substantially greater in skeletal muscle than in blood and is elevated further by dietary nitrate ingestion in human volunteers. We also show that high-intensity exercise results in a reduction in the skeletal muscle nitrate store following supplementation, likely as a consequence of its reduction to nitrite and NO. We also report the presence of sialin, a nitrate transporter, and xanthine oxidoreductase in human skeletal muscle, indicating that muscle has the necessary apparatus for nitrate transport, storage and metabolism. ABSTRACT: Rodent skeletal muscle contains a large store of nitrate that can be augmented by the consumption of dietary nitrate. This muscle nitrate reservoir has been found to be an important source of nitrite and nitric oxide (NO) via its reduction by tissue xanthine oxidoreductase. To explore if this pathway is also active in human skeletal muscle during exercise, and if it is sensitive to local nitrate availability, we assessed exercise-induced changes in muscle nitrate and nitrite concentrations in young healthy humans, under baseline conditions and following dietary nitrate consumption. We found that baseline nitrate and nitrite concentrations were far higher in muscle than in plasma (∼4-fold and ∼29-fold, respectively), and that the consumption of a single bolus of dietary nitrate (12.8 mmol) significantly elevated nitrate concentration in both plasma (∼19-fold) and muscle (∼5-fold). Consistent with these observations, and with previous suggestions of active muscle nitrate transport, we present western blot data to show significant expression of the active nitrate/nitrite transporter sialin in human skeletal muscle. Furthermore, we report an exercise-induced reduction in human muscle nitrate concentration (by ∼39%), but only in the presence of an increased muscle nitrate store. Our results indicate that human skeletal muscle nitrate stores are sensitive to dietary nitrate intake and may contribute to NO generation during exercise. Together, these findings suggest that skeletal muscle plays an important role in the transport, storage and metabolism of nitrate in humans.

Inhaled nebulized nitrite and nitrate therapy in a canine model of hypoxia-induced pulmonary hypertension.

Dysfunction in the nitric oxide (NO) signaling pathway can lead to the development of pulmonary hypertension (PH) in mammals. Discovery of an alternative pathway to NO generation involving reduction from nitrate to nitrite and to NO has motivated the evaluation of nitrite as an alternative to inhaled NO for PH. In contrast, inhaled nitrate has not been evaluated to date, and potential benefits include a prolonged half-life and decreased risk of methemoglobinemia. In a canine model of acute hypoxia-induced PH we evaluated the effects of inhaled nitrate to reduce pulmonary arterial pressure (PAP). In a randomized controlled trial, inhaled nitrate was compared to inhaled nitrite and inhaled saline. Exhaled NO, PAP and systemic blood pressures were continuously monitored. Inhaled nitrite significantly decreased PAP and increased exhaled NO. In contrast, inhaled nitrate and inhaled saline did not decrease PAP or increase exhaled NO. Unexpectedly, we found that inhaled nitrite resulted in prolonged (>5 h) exhaled NO release, increase in nitrate venous/arterial levels and a late surge in venous nitrite levels. These findings do not support a therapeutic role for inhaled nitrate in PH but may have therapeutic implications for inhaled nitrite in various disease states.

Compensatory mechanisms in myoglobin deficient mice preserve NO homeostasis.

The mechanism for nitric oxide (NO) generation from reduction of nitrate (NO3-) and nitrite (NO2-) has gained increasing attention due to the potential beneficial effects of NO in cardiovascular diseases and exercise performance. We have previously shown in rodents that skeletal muscle is the major nitrate reservoir in the body and that exercise enhances the nitrate reduction pathway in the muscle tissue and have proposed that nitrate in muscle originates from diet, the futile cycle of nitric oxide synthase 1 (NOS1) and/or oxidation of NO by oxymyoglobin. In the present study, we tested the hypothesis that lack of myoglobin expression would decrease nitrate levels in skeletal muscle. We observed a modest but significant decrease of nitrate level in skeletal muscle of myoglobin deficient mice compared to littermate control mice (17.3 vs 12.8 nmol/g). In contrast, a NOS inhibitor, L-NAME or a low nitrite/nitrate diet treatment led to more pronounced decreases of nitrate levels in the skeletal muscle of both control and myoglobin deficient mice. Nitrite levels in the skeletal muscle of both types of mice were similar (0.48 vs 0.42 nmol/g). We also analyzed the expression of several proteins that are closely related to NO metabolism to examine the mechanism by which nitrate and nitrite levels are preserved in the absence of myoglobin. Western blot analyses suggest that the protein levels of xanthine oxidoreductase and sialin, a nitrate transporter, both increased in the skeletal muscle of myoglobin deficient mice. These results are compatible with our previously reported model of nitrate production in muscle and suggest that myoglobin deficiency activates compensatory mechanisms to sustain NO homeostasis.

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation.

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) in platelets is increased by nitrite and partially deoxygenated erythrocytes.

Nitrite is recognized as a bioactive nitric oxide (NO) metabolite. We have shown that nitrite inhibits platelet activation and increases platelet cGMP levels in the presence of partially deoxygenated erythrocytes. In this study, we investigated the effect of nitrite on phosphorylation of vasodilator-stimulated phosphoprotein on residue serine 239 (P-VASPSer239), a marker of protein kinase G (PKG) activation, in human platelets. In platelet-rich plasma (PRP), nitrite itself had no effect on levels of P-VASPSer239 while DEANONOate increased P-VASPSer239. Deoxygenation of PRP + erythrocytes (20% hematocrit) raised baseline P-VASPSer239 in platelets. At 20% hematocrit, nitrite (10 µM) increased P-VASPSer239 in platelets about 31% at 10-20 minutes of incubation while the levels of P-VASPSer157, a marker of protein kinase A (PKA) activation, were not changed. Nitrite increased P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes at 20-40% hematocrit, but the effects were slightly greater at 20% hematocrit. In conclusion, our data confirm that nitrite increases P-VASPSer239 in platelets in the presence of deoxygenated erythrocytes. They also further support the idea that partially deoxygenated erythrocytes may modulate platelet activity, at least in part, via the NO/sGC/PKG pathway from NO formed by reduction of circulating nitrite ions.