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Published in final edited form as: Nitric Oxide. 2016 Mar 19;55-56:54–61. doi: 10.1016/j.niox.2016.03.005

Nitrate as a source of nitrite and nitric oxide during exercise hyperemia in rat skeletal muscle

Barbora Piknova 1,1,2, Ji Won Park 1,1, Kai Kwan (Jeff) Lam 1, Alan N Schechter 1
PMCID: PMC4860042  NIHMSID: NIHMS773136  PMID: 27000467

Abstract

The presence of nitric oxide (NO) synthase enzymes, mainly the NOS1 isoform, in skeletal muscle had been well established; however in the last decade it has been realized that NO may also be produced by reduction of nitrate and tissue nitrite. We have recently shown that rodent skeletal muscle contains unusually high concentrations of nitrate, compared to blood and other tissues, likely produced by oxidation of NOS1-produced NO. In the present study we measured nitrate and nitrite levels in Wistar rat leg tissue before and after acute and chronic exercise of the animals on a treadmill. We found a very large decrease of muscle nitrate levels immediately after exercise accompanied by a transient increase of nitrite levels. A significant decrease in blood nitrate levels accompanied the changes in muscle levels. Using skeletal muscle tissue homogenates we established that xanthine oxidoreductase (XOR) is at least partially responsible for the generation of nitrite and/or NO from nitrate and that this effect is increased by slight lowering of pH and by other processes related to the exercise itself. We hypothesize that the skeletal muscle nitrate reservoir contributes significantly to the generation of nitrite and then, probably via formation of NO, exercise-induced functional hyperemia. A model for these metabolic interconversions in mammals is presented. These reactions could explain the muscle-generated vasodilator causing increased blood flow, with induced contraction, exercise, or hypoxia, postulated more than 100 years ago.

Keywords: nitrate, nitrite, nitric oxide, functional hyperemia, exercise, skeletal muscle

Introduction

Nitric oxide (NO) has a very broad range of roles and effects in living organisms. In mammals NO is known to be involved in regulation of vascular tone, blood clotting and immune responses as well as neuronal transmission. It is produced in tissues either by the nitric oxide synthase (NOS) family of enzymes from arginine when oxygen is abundant (1) or by various nonenzymatic reactions from nitrite reduction when oxygen availability is low (2). Nitrite in biological tissues is rapidly oxidized to nitrate by oxy-heme proteins, but it can be slowly converted back to nitrite by bacterial enzymes in the microbiome (3, 4) and by several mammalian tissue nitrate reductases (5). NO itself is also directly oxidized into nitrate by oxy-heme proteins. Nitrate and, to a lesser extend nitrite, in addition to these metabolic conversions, also enters the body through the diet (6). For details on the NO – nitrite – nitrate cycle see (7–9) and the extensive review by van Faassen et al. (10).

Until recently, blood and several internal organs, mainly liver, were the only tissues with known active nitrate/nitrite-to-NO reductase activity (5). Interestingly, in spite of the long known robust expression of functional NOS1 in skeletal muscle (11), this tissue has not received much attention in the nitric oxide field and only limited information exists about the nitric oxide cycle in skeletal muscle. Most of the information is still confined to the muscular dystrophy field, since the observations that loss of NOS1 from the dystrophin protein complex is associated with muscular dystrophies and, recently, likely also with sarcopenia (12, 13).

Functional hyperemia – increased blood flow into active tissue to match increased metabolic demands - had been studied in skeletal muscle during exercise for more than 100 years. The magnitude of hyperemia in exercising skeletal muscle tissue is spectacular – tissue blood flow frequently increases 10 to 20 fold or more – compared to the resting state as the rate of oxygen extraction from blood increases from 20–40% at resting state up to 70–80% during heavy exercise (14). The observed degree of functional hyperemia results from the balance between tonic vasoconstriction in both resting and contracting skeletal muscle, maintained by sympathetic nervous system activity and the counteraction of released vasoactive factors – the phenomenon called functional sympatholysis. Many potential mediators of vasodilation, either blood-born, such as ATP released from red blood cell or angiotensin, or other factors of local tissue origin, such as prostaglandins, have been proposed and studied but none has all the characteristics to accord with major experimental results. Since its discovery, nitric oxide, one of the most potent vasodilators, has also been proposed as an important player in functional hyperemia but inconsistent results with NOS enzyme inhibitors has led to skepticism about this hypothesis (15–17).

Smooth muscle was recognized early as a main target organ for NO in vascular homeostasis maintenance (18); the effects of NO in skeletal muscle have been much less studied. We previously observed that skeletal muscle is a very large nitrate reservoir in the mammalian body, with a strong nitrate gradient from skeletal muscle-to-blood-to-liver in both rats and mice (19). We also demonstrated low but significant levels of nitrate reduction in muscle extracts. These results are consistent with the known expression of functional NOS1 and the presence of large amounts of oxymyoglobin in skeletal muscle and suggest that nitrate/nitrite/NO metabolism may be important in muscle.

We now propose that NO can be generated from the endogenous skeletal muscle nitrate reservoir by nitrate/nitrite reductases residing in skeletal muscle. We develop this idea further considering that the in situ released NO plays an important role in active functional hyperemia. This hypothesis is based on the observation that exercise significantly decreases levels of nitrate and transiently increases levels of nitrite in rat skeletal muscle. Xanthine oxidoreductase residing in skeletal muscle tissue is able to generate nitrite and NO in this tissue; an effect increased by mild acidic conditions, such as caused by exercise-induced lactic acid accumulation and possibly by hypoxia. This simple mechanism involving only skeletal muscle tissue metabolism and independent of reactions in other organs/tissues could at least partially explain how the functional hyperemia in exercising skeletal muscle is affected and maintained over the duration of exercise.

Materials and Methods

Exercise protocol

Adult Wistar rats exercised on a rat treadmill (Columbus Industries, Columbus, OH) at constant speed of 0.32 m/s continuously for 45 min, followed by 60 min of rest and another 45 min of exercise. This 45-60-45 running routine was used in the whole study in two types of experiments. This exercise protocol was empirically developed in our laboratory and was approved for use by NIDDK Animal Care and Use Committee.

First, we were interested how long the single bout exercise effects on nitric oxide metabolism lasted. For this project, animals were allowed to rest for 0, 3, 6 and 12 hours after the end of exercise. At these time points they were euthanized and samples collected and processed as described below in Sample collection and processing.

Second, we were interested in the effects of cumulative exercise on nitric oxide metabolism. For this project, we used the standard 45-60-45 routine for 0, 1, 3, 5 or 7 consecutive days. Immediately after finishing the last exercise, animals were euthanized and blood and tissues collected as described below in Sample collection and processing.

Sample collection and processing

Adult Wistar male rats (n = 50, weight 250 ± 50 g, Charles River Laboratories, Wilmington, MA) were enclosed in an anesthesia box and anesthetized using 5% isoflurane mixture with air. Anesthetized animals were placed on a pad in supine position and anesthesia continued through a nose cone. The thoracic cavity was opened and ~9–10 ml of blood collected by cardiac puncture; representing about two-thirds of total blood volume for an animal of this size. Heparin was used as an anticoagulant in nitrite and nitrate determinations. Immediately after its draw, blood was mixed with a solution containing potassium ferricyanide, NEM and detergent in final ratio 2:1 as described in (20) in order to conserve nitrite from degradation by hemoglobin. Animals were then perfused using heparin-containing saline to flush the remaining blood out of tissues. Perfusion continued until no blood was detected in outgoing saline and liver and kidneys were significantly discolored. Samples from liver and skeletal muscle from the hind legs were then collected and placed into 250 μl of nitrite preserving solution containing potassium ferricyanide for chemiluminescence analysis or flash frozen on dry ice for the Western blot and nitrate or nitrite reductase assays. All samples were stored at −80°C until analysis. Animals were housed in a 12-hour light/dark cycle environment with access to food and drinking water ad libitum. All animal procedures were carried out according to the recommendations in the Guide for the Care and Use of Laboratory Animals of NIH under NIDDK Animal Care and Use Committee approved protocol.

Standard chemiluminescence nitrite and nitrate assays in rat tissues were carried out according to previously published procedures (20, 21). Tissue samples were weighed, mixed with additional “stop” solution and homogenized using GentleMacs (Miltenyi Biotec Inc, Auburn, CA). Proteins were precipitated using methanol (dilution 1:3 sample:methanol) and samples were centrifuged at 11,000 g for 5 min at 4°C to separate most of the protein. Supernatants were collected and used to determine nitrite/nitrate content by chemiluminescence (Sievers 280i Nitric Oxide Analyzer, GE Analytical Instruments, USA).

Nitrate reductase assays in rat skeletal muscle tissues were performed according to the recently published procedures (22). Briefly, tissue was homogenized using GentleMacs tissue dissociator (Miltenyi Biotech, Auburn, CA, USA), total protein in the homogenate was determined using the bicinchoninic acid (BCA) assay kit (Pierce Rockford, IL) and adjusted as necessary to 5 mg/ml. Then 500 μM nitrate together with cofactor mix for nitrate reductases (AO/XOR) was added and aliquots were taken at 0 min, 30 min, 1 h, 2 h, 3 h, 4 h and 24 h and analyzed by chemiluminescence for nitrite content. Cofactor mix consisted of 1 mM NADPH (Fluka), 2 mM UDP glucuronic acid (UDPGA), 0.5 mM glutathione (GSH), 0.5 mM NAD+ and NADH (all from Sigma) in 100 mM phosphate buffer pH 7.4 or pH 6.5 (22). Experiments were performed at 37°C and samples were kept at 2% oxygen. Functional hyperemia in skeletal muscle occurs during intense exercise when oxygen availability in muscle tissue is decreased. Resulting reduced oxygen concentration depends on exercise intensity and duration. We choose 2% oxygen to perform the in vitro experiments, because we hypothesized that it would be close to the reduced oxygen tensions muscle may be subjected to during exercise.

To investigate if the observed nitrate reduction in rat skeletal muscle is a result of action of xanthine oxidoreductase (XOR), a known nitrate reductase, we used oxypurinol, an XOR inhibitor. 100 μM oxypurinol was added to the tissue homogenate together with cofactor mixture. Nitrate at a concentration of 300 μM was used to initiate the reaction and the assay proceeded as described earlier.

Nitrite reduction into nitric oxide in rat skeletal muscle tissue was measured using chemiluminescence. Skeletal muscle homogenate (5mg/ml) was incubated with cofactor mixture in 100mM phosphate buffer pH 6.5 for 5 min prior to nitrite (1mM or 2mM) addition. For inhibition of XOR, 100 μM oxypurinol was added together with the cofactor mixture. Nitric oxide generation was monitored for 20 min at room temperature.

Rat skeletal muscle homogenates were prepared by GentleMacs tissue dissociator with RIPA buffer (Sigma, Cat.# R0278) containing protease inhibitor cocktail (Sigma, Cat.# S8830), and then protein concentration was determined by BCA assay. Denatured samples (50 μg) were run on SDS–PAGE and then transferred to nitrocellulose membrane. The membrane was incubated with primary antibodies (anti-xanthine oxidase: Santa Cruz Biotechnology, SC-20991, anti-GAPDH: Sigma-Aldrich, G9545) overnight at 4°C, then immunoblotted with secondary antibodies (L icor Biosciences, 926-32211, 926-68070) for 1 hour at 4°C. The blots were imaged using the Odyssey imaging system (Licor Biosciences).

Statistical significance of results was tested using the one way ANOVA test.

Results

Figures 1A and B show levels of nitrate and nitrite ions in liver, blood and skeletal muscle of sedentary and exercised Wistar rats and are very similar to our recent publication in which we reported that skeletal muscle in resting rodents have high levels of nitrate (19). To examine possible changes in these species with exercise, nitrate and nitrite levels were measured after the animals completed one exercise session which consisted of two 45 min runs on a treadmill with a 60 min rest period between runs, as described above. They were euthanized at four different time points after exercise completion – 0, 3, 6 and 12 hours - and tissue and blood samples were prepared, flash frozen and analyzed. Results from the exercising rats, together with sedentary controls, are shown in Figure 1A (nitrate) and 1B (nitrite). Black bars are non-exercised controls (n=14), red, green, purple and blue bars represent results from animals immediately after the exercise completion (n=6), after 3 hours (n = 6), after 6 hours (n = 4) and after 12 hours (n = 4) of rest after exercise, respectively.

Figure 1.

Figure 1

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Effect of acute exercise on nitrate (A) and nitrite (B) levels in liver, blood and skeletal muscle of Wistar rats. Exercise session consisted of two 45 min run on a treadmill with 60 min rest period between runs. Samples were prepared immediately after the end of exercise session (0 hours, red bar, n = 6), 3 (green bar, n = 6), 6 (purple bar, n = 4) and 12 hours (blue bar, n = 4) after exercise. Results are compared with sedentary controls (black bar, n = 14).

Figure 1A shows that immediately after finishing the exercise, nitrate levels in muscle declines to about 30% of the controls and at three hours after the exercise we found about a 20-fold depletion of nitrate compared to controls. Blood levels follow a similar pattern but the relative decline is perhaps slightly more rapid. Liver tissue has a transient increase in nitrate and then a fall to less than half of those measured in control values. In contrast, muscle nitrite levels increase about three-fold immediately after exercise and then decline to about two-fold the sedentary controls (Figure 1B). Exercise or the following resting periods do not change levels of nitrite in liver or blood, as also shown in Figure 1B.

We repeated these experiments on a more chronic basis with animals being exercised in a similar way in two sequential sessions each day and then immediately killed after 1, 3, 5 or 7 consecutive days of such exercise periods. Data are shown for 1day (red bars, n= 6), 3 days (green bars, n = 6), 5 days (blue bars, n = 4) and 7 days (grey bars, n = 4) as well as sedentary controls (black bars, n = 14).

Figure 2A shows the exercise dose dependency effect on levels of nitrate in liver, blood and skeletal muscle. Exercise influences levels of nitrate in all three organs studied. Depletion from skeletal muscle is quite dramatic – an initial 3-fold drop after one day is followed by 6.5-, 10- and 12.5-fold decreases after 3, 5 and 7 days of exercise.

Figure 2.

Figure 2

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Effect of repetitive exercise on nitrate (A) and nitrite (B) levels in liver, blood and skeletal muscle of Wistar rats. Animals exercised for 1 (red bar, n= 6), 3 (green bar, n = 6), 5 (blue bar, n = 4) and 7 (grey bar, n = 4) consecutive days, every exercise session consisted of two 45 min run on treadmill with 60 min rest period between runs. Results are compared with sedentary controls (black bar, n = 14).

At the first exercise day, when compared to the sedentary controls, there is a significant 3-fold increase in liver nitrate and a 3.5-fold decrease in blood levels. The increase in the liver persists through the whole exercise regimen, even though levels of nitrate detected after 3, 5 and 7 days of exercise are not as much elevated as after the first day, while blood levels do not show further statistically significant changes. However, as shown in Figure 2B, exercise does not change significantly nitrite levels in liver or blood, regardless of its duration. However, when compared to the sedentary control, there is a significant 3-fold increase of nitrite concentration in the skeletal muscle after the first exercise session. These levels of nitrite in muscle continued to diminish and there was no significant difference between nitrite at baseline and levels measured after 5 or 7 days of exercise.

These results, both those in Figures 1 and 2, suggest that the strong depletion of muscle nitrate with exercise involves its conversion to nitrite and then further metabolism, likely to NO or its metabolic products. To further elucidate the possible fate of nitrate in skeletal muscle we performed nitrate reductase assays in skeletal muscle homogenates from control sedentary rats and from rats after different periods of exercise. Results are shown in Figure 3A–C. In panel A, we compare possible effects of decreased pH and one day of exercise on nitrate reduction in rat skeletal muscle homogenates. As can be seen, slightly acidic conditions (pH 6.5) increased 1.7-fold the total amount of measured nitrite from 500μM of added nitrate compared with neutral conditions (pH 7.4) (black diamonds vs gray diamonds) in control sedentary rats. However, this effect does not fully account for the total effect of about a 3.4-fold nitrite increase observed in the homogenates after one day of exercise (red squares vs. grey diamonds). Panel B shows the time course of nitrite generation from 500μM added nitrate in tissue homogenates from control animals (black diamonds) and animals after 1, 3 and 5 days of exercise at pH 6.5 (red squares, green triangles and blue circles, respectively). Experiments were performed at pH 6.5 to better mimic acidic conditions in working muscle, which is mainly due to accumulated lactic acid. In all cases nitrite steadily increased in tissue homogenates over the period of two hours. When compared with controls, there was a significant 2-fold increase of the amounts of nitrite generated by tissue homogenates from rats after completing one day of exercise. There was no significant difference in the amount of nitrite generated by tissue homogenates from rats exercising for 3 and 5 days when compared to control animals.

Figure 3.

Figure 3

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Nitrate reductase activity of skeletal muscle homogenate in control Wistar rat and after exercise. A: Comparison of the effect of decreased pH and 1 day exercise on nitrate reduction in rat skeletal muscle homogenate. Gray diamonds – control, pH 7.4 (n=3), black diamonds – control, pH 6.5 (n=4), red squares – 1 day exercise, pH 6.5 (n=4). Nitrite was generated from 500μM added nitrate. B: Time course of nitrate to nitrite conversion after addition of 500μM nitrate to skeletal muscle homogenate in sedentary animals (black diamonds), after 1 day (red squares), 3 days (green triangles) and 5 days (blue circles) of exercise. Four rats were used in each case. C: Inhibition of nitrate reduction in tissue homogenate by addition of oxypurinol, xanthine oxidoreductase inhibitor. Black diamonds– tissue homogenate from rats after 1 day exercise, red squares – homogenate from tissue after 1 day exercise with addition of 100μM oxypurinol. Presented data are average of results obtained from three different rats. Nitrite was generated from 500μM added nitrate.

Panel C of Figure 3 documents that nitrate reduction in skeletal muscle homogenates from rats exercising for one day can be stopped by addition of 100μM of oxypurinol, a XOR inhibitor.

We also measured XOR protein by Western Blots but found no changes with the duration of exercise (results not shown).

In Figure 4 we show that skeletal muscle also contains all necessary components to generate significant amount of NO from nitrite. Panel A shows the time course of NO generation from 1mM and 2mM nitrite over the time period of 20 min in control homogenates from sedentary rats and after one day of exercise. These results are quantified in panel B as total amount of NO in pmoles released per mg tissue per minute. The NO generation is dose-dependent and there is 1.5-fold more NO produced in tissue homogenates from exercising rats when compared with control sedentary rats. NO generation from nitrite can be inhibited by addition of 100μM oxypurinol, as shown by the green bars.

Figure 4.

Figure 4

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Nitric oxide (NO) generation from skeletal muscle homogenate after nitrite addition in control rats and after 1 day of exercise. A: Time course of NO generation by tissue homogenate - comparison of addition of 1mM and 2mM nitrite. Control after addition of 1mM (black line, a) or 2mM (gray line, b) nitrite, 1 day exercise after addition of 1mM (red line, c) or 2mM (blue line, d) nitrite. B: Quantification of NO generation based on the area under curves for each treatment (calculated using Origin Software). Black bars represent data from sedentary controls, red bars are results from tissues collected from animals after exercising for 1 day. Green bars represent nitric oxide generation in the presence of 100μM oxypurinol (an inhibitor of xanthine oxidoreductase) from tissues of animals exercised for 1 day. Gray bars are nitric oxide production in the presence of L-NAME (a NOS inhibitor) from animals exercised for 1 day.

Figure 5 shows levels of nitrate (A) and nitrite (B) in liver, blood and skeletal muscle of sedentary animals subjected to intake of L-NAME in drinking water (1g/L, NOS inhibitor, gray bars) for 7 consecutive days as compared to control animals (black bars). As seen in Panel A, NOS inhibition alters measured nitrate content in these tissues significantly. NOS inhibition lowers nitrate greatly in blood and skeletal muscle – we observed about a 5-fold decrease in these tissues compared to controls – but raises liver levels slightly (an unexplained effect we have seen in various organs in other rodent studies). As seen in Panel B, seven days of NOS inhibition with L-NAME causes only slight variations of steady state nitrite levels in liver, blood or muscle.

Figure 5.

Figure 5

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Effect of L-NAME treatment on nitrate (A) and nitrite (B) levels in liver, blood and skeletal muscle of Wistar rats. Animals were on control water (black bar, n=3) or 1g/L of L-NAME water (grey bar, n = 3) for 7 consecutive days.

Discussion

In our previous study we have shown that skeletal muscle is an endogenous nitrate reservoir, perhaps the largest one in the mammalian body (19). Presumably, nitrate stored in this tissue originates from NO formed from arginine by NOS1 and its oxidation by muscle oxymyoglobin. The first hypothesis is supported by the facts that muscle levels are much higher than blood levels and by the large decrease of nitrate levels in skeletal muscle, after NOS inhibition by L-NAME, data we reported in that study. In the current study, we hypothesized that nitrate stored in the skeletal muscle could be rapidly converted back to NO, via a nitrite intermediate, by mammalian nitrate reductase system(s) and used to regulate blood flow through the muscle in situations of increased oxygen consumption, such as during exercise.

The relationship of muscle blood flow to exercise, induced muscle contractions, or decreased oxygen supply has been studied since the mid-19th century. This functional hyperemia is physiologically quite complex as increases in blood flow, which are usually up to 20-fold, but can reach 100-fold normal values, must involve the entire circulatory system to maintain overall blood circulation. Thus changes in cardiac function, the autonomic and central nervous systems, as well as peripheral resistance are all involved in the systemic response (23). However, the very large changes in muscle suggest that this organ has major local mechanisms for changing vasomotor tone. Indeed as early as 1880 Gaskell (24), and later in 1928 Bernheim and Dixon (25) proposed that some substances produced by muscle accounts for the hyperemia and various physiologists since then have revisited this idea.

However, despite the study of several dozen possible vasodilators none has had all the properties necessary to account for the bulk of the many experimental results with various animal models, as well as human studies. The discovery of NO as EDRF, a very potent vasodilator, led to many studies testing whether this short-lived molecule could account for much of functional hyperemia. Most such studies were based on inhibition of the NOS enzymes and the results led largely to the conclusion that NO was not the long sort vasodilator substance (17).

The realization in the last decade that much NO, especially that produced under hypoxic conditions, was a result of reduction of nitrite and possibly nitrate in the mammalian body, rather than synthesis from arginine by the NOS enzymes, led us to hypothesize that these reductive processes could explain the formation of such local vasodilators. Our recent finding of very high skeletal muscle levels of nitrate ions caused us to test its role in the response of rodents to exercise.

In the experiments presented on Figures 1A and B we determined that changes in nitrate and nitrite levels are exercise-related and occur mainly while the muscle is exercising – the major decreases in blood and muscle nitrate levels and the spike in skeletal muscle nitrite are observed in tissues extracted immediately after acute exercise, Further decreases in both nitrite and nitrate with time of rest was observed. Two factors can play the role in early nitrite/nitrate decay after exercise. We assume that nitrate is being used to produce nitrite, which is then consumed in the muscle tissue or is transported in the body; however, other mechanisms could be envisioned such as those involving active transport (see below). Figures 2A and B show nitrate and nitrite concentrations after different degrees of exercise – 0, 1, 3, 5 and 7 consecutive days of exercise, a more chronic experiment. The overall results are very similar to those we observed in the more acute experiments cited above.

We have shown previously that skeletal muscle homogenate is able to reduce exogenously added nitrate into nitrite and that xanthine oxidoreductase is responsible for at least part of this reduction (19). Indeed, in 1928 Bernhein and Dixon (25) reported nitrate reduction into nitrite by muscle extracts. Figure 3B shows results of nitrate reductase assays in muscle tissue homogenates as a function of exercise dose. Interestingly, the highest rate of nitrate reduction occurs in muscle immediately after one day of exercise. Xanthine oxidoreductase again appears to be the major enzyme responsible for the observed nitrate reduction as the reaction can be completely abolished by addition of oxypurinol, a xanthine oxidoreductase inhibitor, as documented in Figure 3C. There is strong experimental evidence for this reaction in liver, as shown first by Zweier’s group (2) and then by Jansson et al (22), due to the substantial amount of xanthine oxidoreductase in liver tissue.

During exercise, skeletal muscle tissue progressively accumulates lactic acid and becomes acidic (26, 27). Xanthine oxidoreductase activity is highly pH-dependent and its activity increases at acidic conditions (2), consistent with the data in Figure 3A where nitrate reduction in control sedentary rat tissue is much higher at pH 6.5 than at pH 7.4. However, this pH drop does not account completely for the high increase of nitrate reduction observed in animals after a single day of exercise (pH 6.5) and suggests other mechanisms influencing nitrate reduction occur in muscle tissue as a result of exercise. Indeed, Millar and coworkers reported general ability of mammalian (milk) XOR to act as the nitrate/nitrite reductase was increased under hypoxic conditions (28). Recently, Cantu-Mendellin and Kelley reevaluated XOR as a nitrite/nitrate reductase under hypoxia (29).

Although changes in formation and consumption of nitrate and nitrite with exercise is the most likely explanation of our overall results, we cannot exclude effects of active transport mechanisms, especially that of nitrate. Currently, there are only two known protein systems that had been shown to transport nitrate ions in mammalian cells – sialin (30) and aquaporin 6 (31), although neither is well characterized. We performed Western blot of rat skeletal muscle and found that sialin is present in this tissue but exercise did not change its amount (data not shown), but this does not exclude the possibility of exercise-induced increase of transport activity. Literature search did not reveal if aquaporin 6 is present or absent from skeletal muscle tissue. An excretion pathway, via the kidney and aquaporin 6 has been reported, but we have not yet carefully studied levels of nitrate and nitrite in urine.

We next asked if the nitrite found in skeletal muscle can be further reduced to NO, one of the most powerful vasodilators in body. We again used tissue homogenates but added nitrite instead of nitrate and NO formation was measured using chemiluminescence (Figure 4A and B). In order to be detected we needed about 1000-times excess of nitrite when compared with our measurements of transient levels – however our own values of these transients may be far lower than the actual muscle maxima. Further, we consider this experiment as a proof of principle and do not draw any quantitative conclusions about the amount of NO generated at physiological conditions (e.g. relative hypoxia) by muscle tissue. Addition of oxypurinol abolishes most NO generation, and is evidence in support of the existence of nitrate reduction to nitrite and of xanthine oxidoreductase being the major enzyme carrying out this reduction process, as well as nitrate reduction to nitrite. The residual NO formed after oxypurinol addition could be from direct synthesis by the NOS1 enzymes present in skeletal muscle. According to our calculations for the model system, there is around 1 pmole of NO generated per mg of tissue per minute from 2 mM added nitrite. When taking into account that 1 pmole of NO is generated from ~1 μmole of nitrite per minute, this rough calculation leads to the estimate of 10−6 fraction of nitrite being converted into NO per minute. This may reflect that the homogenate system is not optimized for this reaction, but even if the amount of NO generated from the physiologically relevant levels of nitrite in muscle is low, it still could sustain exercise-induced hyperemia either by itself or in combination with other released active factors. It should also be noted that nitrite itself may act as a vasodilator or support the release of other active substances – like ATP from red blood cells (32).

In our previous study (19) we hypothesized that colocalization of functional NOS, present in skeletal muscle in large quantities, and oxymyoglobin in muscle cell leads to the oxidation of NO to nitrate and to the accumulation of nitrate in the muscle tissue. In Figure 5 we show the results of measurements of nitrate (A) and nitrite (B) levels in skeletal muscle of sedentary rats after seven days at inhibition of NOS-mediated NO formation and with no intervention. Inhibition of NO formation by L-NAME leads to a 5-fold decrease of nitrate measured in skeletal muscle and only very small effect on nitrite levels in these tissues. These results are in agreement with the hypothesis that the large nitrate reservoir in skeletal muscle is primarily a result of accumulation of nitrate formed by oxidation of NO formed in the skeletal muscle by oxymyoglobin. We are testing this hypothesis further with myoglobin knock-out mice.

In Figure 6 we present a model for the metabolic conversions of nitrogen oxides in muscle that we postulate as important in functional hyperemia in context of other known mammalian tissue reactions of these molecular species. In the lower left box we illustrate, for muscle tissue, the constant formation of NO by NOS1 and its reaction (along with any endothelial generated NO that survives long enough to diffuse into muscle tissue) with oxymyoglobin to form nitrate. We postulate that exercise causes rapid reduction of the stored nitrate to nitrite and then rapid formation of NO by the mechanisms shown, as well as other mechanisms related to the exercise, such as partial hypoxia, that requires further study. This NO could dilate the muscle blood vessels and increase flow; however it is likely that upstream effects also result from exercise, mediated by the central or autonomic nervous systems, or possibly effects of NO on neurons within the muscle bundles themselves, as total blood flow to the muscle must also be augmented and blood pressure maintained.

Figure 6.

Figure 6

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Model of the complete nitrate-nitrite-NO cycle in mammalian body. Nitrate and nitrite fluxes between different compartments: skeletal muscle, blood, liver (representative of internal organs) and endothelium are depicted as arrows, together with important proteins/enzymes responsible for the conversions. There is also an important flux of nitrate and nitrite from diet into the blood. The nitrate reductases of microbiome reduce significant amount of nitrate into nitrite.

Figure 6 also shows the metabolic pathways of NO in endothelial cells, blood and liver that have been studied extensively in the last decade and would also contribute to overall fluxes and levels of these compounds in the muscle tissue, probably via passive diffusion. It should be noted that at present we do not know the possible roles of nitrate reduction in other muscle forms, or other tissues in which these parameters have not been extensively studied. Clearly this is a complex system with many feedback loops and cannot be quantitated very easily. It will also be noted that this model returns NO to a prominent role in functional muscle hyperemia that was anticipated with its characterization in the 1980’s but was largely discarded on the basis of short-term studies with inhibitors of the NOS enzymes. It is also consistent with the idea, dating at least to 1880, that muscle generated substances have major effects on its own blood flow, what is now designated an autocrine system.

Highlights.

  • Skeletal muscle - the largest indigenous nitrate reservoir in mammalian body.

  • Exercise depletes nitrate and transiently increases nitrite in skeletal muscle.

  • Skeletal muscle xanthine oxidoreductase is able to reduce nitrate to nitric oxide.

  • Nitrate reduction to NO – a new pathway for active hyperemia in skeletal muscle?

Acknowledgments

Authors would like to thank Dr Mark StClair for his advices and help with animal protocol and work.

Footnotes

Competing financial interests.

BP, JWP and JL declare no conflict of interest. ANS is listed as a co-inventor on several patents issued to the National Institutes of Health for the use of nitrite salts for the treatment of cardiovascular diseases. He receives royalties based on NIH licensing of these patents for clinical development but no other compensation. These arrangements do not affect his adherence to Nitric Oxide journal policies.

Contributions.

BP, JWP and ANS designed the experiments and wrote the manuscript. BP and JWP performed the research and analyzed the data. JL was in charge of exercising animals and collected tissue and blood samples. All authors contributed to data interpretation and commented on manuscript.

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