Endogenous Nitrate Research Articles

A New Real-Time Simple Method to Measure the Endogenous Nitrate Reductase Activity (Nar) in Paracoccus denitrificans and Other Denitrifying Bacteria.

The transmembrane nitrate reductase (Nar) is the first enzyme in the dissimilatory alternate anaerobic nitrate respiratory chain in denitrifying bacteria. To date, there has been no real-time method to determine its specific activity embedded in its native membrane; here, we describe such a new method, which is useful with the inside-out membranes of Paracoccus denitrificans and other denitrifying bacteria. This new method takes advantage of the native coupling of the endogenous NADH dehydrogenase or Complex I with the reduction of nitrate by Nar through the quinone pool of the inner membranes of P. denitrificans. This is achieved under previously reached anaerobic conditions. Inner controls confirming the specific Nar activity determined by this new method were made by the total inhibition of the Nar enzyme by sodium azide and cyanide, well-known Nar inhibitors. The estimation of the Michaelis-Menten affinity of Nar for NO3- using this so-called Nar-JJ assay gave a Km of 70.4 μM, similar to previously determined values. This new Nar-JJ assay is a suitable, low-cost, and reproducible method to determine in real-time the endogenous Nar activity not only in P. denitrificans, but in other denitrifying bacteria such as Brucella canis, and potentially in other entero-pathogenic bacteria.

The specificity of endogenous fatty acid nitration: only conjugated substrates support the in vivo formation of nitro-fatty acids

Through multiple pathways, nitrogen dioxide (•NO2) is the main species involved in endogenous nitration reactions. Early studies in the field primarily explored tyrosine nitration, a dominant reaction in the field. It was later shown that lipids are also nitration targets and generate an array of reaction products. Conjugated fatty acids are the preferential substrates of lipid nitration in vivo, generating electrophilic nitro-fatty acids (NO2–FAs), which serve as pleiotropic signaling modulators. In contrast, exposure of bisallylic fatty acids, including linoleic, linolenic and arachidonic acid, to •NO2 does not lead, under biological conditions, to the formation of nitrated species. This review focuses on the reaction mechanisms and products of lipid nitration and substrate specificity, focusing on the differential reactivity of conjugated dienes and bisallylic alkenes.

Protein nitration in the artery wall: A contributor to cardiovascular disease?

Nitration is a well-established post-translational modification of selected free amino acids, as well as proteins, lipids and nucleic acids. Considerable evidence is now available for the formation of long-lived species containing an added –NO2 function on the aromatic rings of tyrosine (Tyr) and tryptophan (Trp) residues (both free and on proteins), to purine nucleobases (and particularly guanine), and to unsaturated lipids within biological systems. Multiple potential mechanisms that give rise to these nitrated species have been identified including reactions of the potent oxidant and nitrating species peroxynitrous acid/peroxynitrite (ONOOH/ONOO−) and via oxidative reactions of heme proteins/enzymes (e.g. peroxidases) with the biologically-relevant anion nitrite (NO2−). •NO2 is likely to be a key intermediate, though involvement of HNO2, NO2+ and NO2Cl has also been proposed. The resulting nitrated products have been widely employed as qualitative or quantitative biomarkers of nitration events in vitro and in vivo. Increasing evidence suggests that at least some of these products are not benign species, with evidence for pro-inflammatory actions. In this article the mechanisms and role of nitration, and particularly that on proteins within the artery wall, in cardiovascular disease is discussed, together with emerging data suggesting that low levels of nitration occur within biological systems in the absence of added oxidants. Both stimulated and endogenous nitration may play a role in modulating cell signaling, alter the structure and function of both cellular- and extracellular proteins, and contribute to various inflammatory pathologies, including atherosclerosis.

Identification and quantification of protein nitration sites in human coronary artery smooth muscle cells in the absence and presence of peroxynitrous acid/peroxynitrite

Peroxynitrous acid/peroxynitrite (ONOOH/ONOO−) is a powerful oxidizing/nitrating system formed at sites of inflammation, which can modify biological targets, and particularly proteins. Here, we show that multiple proteins from primary human coronary artery smooth muscle cells are nitrated, with LC-MS peptide mass mapping providing data on the sites and extents of changes on cellular and extracellular matrix (ECM) proteins. Evidence is presented for selective and specific nitrations at Tyr and Trp on 11 cellular proteins (out of 3668, including 205 ECM species) in the absence of added reagent ONOOH/ONOO−, with this being consistent with low-level endogenous nitration. A number of these have key roles in cell signaling/sensing and protein turnover. With added ONOOH/ONOO−, more proteins were modified (84 total; with 129 nitrated Tyr and 23 nitrated Trp, with multiple modifications on some proteins), with this occurring at the same and additional sites to endogenous modification. With low concentrations of ONOOH/ONOO− (50 μM) nitration occurs on specific proteins at particular sites, and is not driven by protein or Tyr/Trp abundance, with modifications detected on some low abundance proteins. However, with higher ONOOH/ONOO− concentrations (500 μM), modification is primarily driven by protein abundance. ECM species are major targets and over-represented in the pool of modified proteins, with fibronectin and thrombospondin-1 being particularly heavily modified (12 sites in each case). Both endogenous and exogenous nitration of cell- and ECM-derived species may have significant effects on cell and protein function, and potentially be involved in the development and exacerbation of diseases such as atherosclerosis.

Complete nitrogen removal from low-strength wastewater via double nitrite shunt coupling anammox and endogenous nitrate respiration: Functional metabolism and electron transport

Autotrophic nitrogen removal processes based on anammox are facing huge challenges of high effluent quality in wastewater treatment. In this study, a novel hybrid system of anammox coupled with heterotrophic endogenous metabolisms was developed in a single-stage bioreactor to treat real domestic wastewater. Complete nitrogen removal was achieved at a low carbon to nitrogen (C/N) ratio of 2.8 and more than 95.2% nitrogen removal was maintained under an extremely low C/N ratio of 1.9. The nitrite shunt supplied by dual partial pathways (partial nitritation and endogenous partial denitratation) guaranteed the anammox activity with a high contribution to nitrogen removal above 60%. While endogenous denitrification (ED) and denitrifying phosphorus removal (DPR) both participated in nitrate respiration, contributing more than 28% and 9% to nitrogen removal, respectively. The coexistence and enrichment of anammox bacteria (Ca. Brocadia, 0.9%), glycogen accumulating organisms (Ca. Competibacter, 6.4%) and phosphorus accumulating organisms (Dechloromonas, 3.9% and Ca. Accumulibacter, 0.7%) contributed to the co-metabolism of carbon, nitrogen and phosphorus. The functional genes encoding the above metabolism were highly correlated with the dominant strains, indicating that the multiple pathways of carbon and nitrogen metabolism supported the continuous supply of electrons required for nitrogen removal. This study puts forward a novel complete nitrogen removal process by successfully coupling autotrophic and endogenous heterotrophic metabolism.

Insights into integration of polyhydroxyalkanoates (PHAs) production into wastewater treatment: Comparison of different electron acceptors on system function and PHA-producer enrichment

Mixed microbial culture polyhydroxyalkanoates (MMC PHA) process combined with mainstream wastewater treatment is a competitive resource recovery integrated process in which economical non-oxygen electron acceptors can be used to enrich PHA-producer and reduce operating costs. Oxygen and three non-oxygen electron acceptors (endogenous nitrate, exogenous nitrate, and nitrite) were used and comprehensively compared in terms of PHA production ability and microbial community ecophysiology in this study. The results showed that the PHA production ability with exogenous nitrate was comparable to that of oxygen, which had a maximum PHA content of 64.0 wt% (versus 62.3% of oxygen), qPHA of 0.7 mg COD/mg Xa/h, as well as 99.1% denitrification efficiency. The non-oxygen acceptors delayed the time for Arcobacter to occupy the dominant niche in the community, and the environmental selection pressure produced was slightly lower than that of oxygen. The symbiotic community between denitrifying bacteria and PHA-producers ensured the high PHA production potential and denitrification efficiency of the non-oxygen system. An economic assessment showed that the integrated PHA production and nitrate removal process with the mainstream wastewater treatment process proposed in this study could reduce aeration energy by 64%, and PHA recovery yield was 0.1 kg CODPHA/kg CODWAS from waste activated sludge (WAS).

Burkholderiaceae Are Key Acetate Assimilators During Complete Denitrification in Acidic Cryoturbated Peat Circles of the Arctic Tundra.

Cryoturbated peat circles (pH 4) in the Eastern European Tundra harbor up to 2 mM pore water nitrate and emit the greenhouse gas N2O like heavily fertilized agricultural soils in temperate regions. The main process yielding N2O under oxygen limited conditions is denitrification, which is the sequential reduction of nitrate/nitrite to N2O and/or N2. N2O reduction to N2 is impaired by pH < 6 in classical model denitrifiers and many environments. Key microbes of peat circles are important but largely unknown catalysts for C- and N-cycling associated N2O fluxes. Thus, we hypothesized that the peat circle community includes hitherto unknown taxa and is essentially unable to efficiently perform complete denitrification, i.e., reduce N2O, due to a low in situ pH. 16S rRNA analysis indicated a diverse active community primarily composed of the bacterial class-level taxa Alphaproteobacteria, Acidimicrobiia, Acidobacteria, Verrucomicrobiae, and Bacteroidia, as well as archaeal Nitrososphaeria. Euryarchaeota were not detected. 13C2- and 12C2-acetate supplemented anoxic microcosms with endogenous nitrate and acetylene at an in situ near pH of 4 were used to assess acetate dependent carbon flow, denitrification and N2O production. Initial nitrate and acetate were consumed within 6 and 11 days, respectively, and primarily converted to CO2 and N2, suggesting complete acetate fueled denitrification at acidic pH. Stable isotope probing coupled to 16S rRNA analysis via Illumina MiSeq amplicon sequencing identified acetate consuming key players of the family Burkholderiaceae during complete denitrification correlating with Rhodanobacter spp. The archaeal community consisted primarily of ammonia-oxidizing Archaea of Nitrososphaeraceae, and was stable during the incubation. The collective data indicate that peat circles (i) host acid-tolerant denitrifiers capable of complete denitrification at pH 4–5.5, (ii) other parameters like carbon availability rather than pH are possible reasons for high N2O emissions in situ, and (iii) Burkholderiaceae are responsive key acetate assimilators co-occurring with Rhodanobacter sp. during denitrification, suggesting both organisms being associated with acid-tolerant denitrification in peat circles.

Advanced nitrogen removal without addition of external carbon source in an anaerobic/aerobic/anoxic sequencing batch reactor.

Advanced nitrogen removal without the addition of external carbon source is challenging in the conventional biological nitrogen removal processes. This study presented a novel anaerobic/aerobic/anoxic sequencing batch reactor (A/O/A SBR) based on endogenous nitrate (NO3--N) respiration to enhance nitrogen removal. The mean effluent total nitrogen (TN) in the A/O/A SBR could be reduced to as low as 3.5mg/L, when the average influent TN and chemical oxygen demand (COD) were 52.7 and 235.4mg/L, respectively. This advanced nitrogen removal was attributed to the post-denitrification, since 82.7% of TN removal was achieved in the post-anoxic stage. The post-denitrification rate with nitrite (NO2--N, 0.59mg NO2--N/gMLVSS/h) was higher than that with NO3--N (0.35mg NO3--N/gMLVSS/h). Therefore, the post-anoxic time could be further optimized by achieving denitrification via NO2--N. The A/O/A SBR has good potential in achieving advanced nitrogen removal, especially in nitrogen-sensitive rural areas.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

Managing the power grid: how myoglobin can regulate PO2 and energy distribution in skeletal muscle.

An inducible expression system in the alga Nannochloropsis gaditana controlled by the nitrate reductase promoter

The marine microalgal species Nannochloropsis gaditana has emerged as a model organism for research into algal-derived biofuels due to its ability to produce large amounts of lipids. In addition, this species has biotechnological potential owing to its ease of transformation and availability of a sequenced and annotated genome. The ability to control expression of transgenes in an inducible manner is an important requirement for many genetic engineering approaches and as such, inducible promoters are an important component of the molecular toolkit for a host organism. We designed an expression vector containing a ~ 1.3 kb region upstream of the endogenous nitrate reductase gene of N. gaditana and demonstrated this is capable of promoting the expression of a heterologous enhanced green fluorescent protein (eGFP) reporter. In the presence of ammonium, expression of the eGFP reporter was undetectable; however, in the presence of nitrate, eGFP expression is induced. In addition, we demonstrated how altering the nitrogen source of the growth media can be used to precisely control expression. We have shown that the nitrate reductase promoter can be used as a powerful molecular tool for heterologous protein expression in N. gaditana, further contributing to the development of this species as a candidate strain for biotechnology.

Effect of dietary nitrate levels on nitrate fluxes in rat skeletal muscle and liver

Rodent skeletal muscle has high levels of nitrate ions and this endogenous nitrate reservoir can supply nitrite/nitric oxide (NO) for functional hyperemia and/or for other physiological processes in muscle during exercise. Mice with a NOS1 knockout have markedly reduced muscle nitrate levels, suggesting NO production by NOS and its reaction with oxymyoglobin as a source of nitrate. However, oxygen levels are normally low in most internal organs, which raises the possibility that nitrate-derived NO pathway is physiologically important even at “normoxia”, and muscle nitrate reservoir is the main endogenous NO backup when exogeneous (dietary) nitrate intake is low. Using dietary nitrate manipulations, we explore the importance of diet for maintaining and renewal of muscle nitrate reservoir and its levels in other tissues. We found that skeletal muscle nitrate is extensively used when nitrate in diet is low. One week of nitrate starvation leads to dramatic nitrate depletion in skeletal muscle and a substantial decrease in liver. Nitrate depleted from skeletal muscle during starvation is quickly recovered from new dietary sources, with an unexpected significant “overload” compared with animals not subjected to nitrate starvation. Our results suggest the importance of dietary nitrate for nitrate reserves in muscle and in other tissues, when compared with endogenous NOS-derived sources. This requires an active transport mechanism for sequestering nitrate into cells, stimulated by lack of dietary nitrate or other enzymatic changes. These results confirm the hypothesis that muscle is a major storage site for nitrate in mammals.

Determination of endogenous concentrations of nitrites and nitrates in different types of cheese in the United States: method development and validation using ion chromatography

ABSTRACTNitrites and nitrates can be present in dairy products from both endogenous and exogenous sources. In the European Union (EU), 150 mg kg–1 of nitrates are allowed to be added to the cheese milk during the manufacturing process. The CODEX General Standard for Food Additives has a maximum permitted level of 50 mg kg–1 residue in cheese, while in the United States (U.S.) nitrates are unapproved for use as food additives in cheese. In order to be able to investigate imported cheeses for nitrates intentionally added as preservatives and the endogenous concentrations of nitrates and nitrites present in cheeses in the U.S. marketplace, a method was developed and validated using ion chromatography with conductivity detection. A market sampling of cheese samples purchased in the Washington DC metro area was performed. In 64 samples of cheese, concentrations ranged from below the method detection limit (MDL) to 26 mg kg–1 for nitrates and no concentrations of nitrites were found in any of the cheese samples above the MDL of 0.1 mg kg–1. A majority of the samples (93%) had concentrations below 10 mg kg–1, which indicate the presence of endogenous nitrates. The samples with concentrations above 10 mg kg–1 were mainly processed cheese spread, which can contain additional ingredients often of plant-based origin. These ingredients are likely the cause of the elevated nitrate concentrations. The analysis of 12 additional cheese samples that are liable to the intentional addition of nitrates, 9 of which were imported, indicated that in this limited study, concentrations of nitrate in the U.S.-produced cheeses did not differ from those in imported samples.

Development of a sandwich ELISA with potential for selective quantification of human lactoferrin protein nitrated through disease or environmental exposure.

Lactoferrin (LF) is an important multifunctional protein that comprises a large fraction of the protein mass in certain human fluids and tissues, and its concentration is often used to assess health and disease. LF can be nitrated by multiple routes, leading to changes in protein structure, and nitrated proteins can negatively impact physiological health via nitrosative stress. Despite an awareness of the detrimental effects of nitrated proteins and the importance of LF within the body, cost-effective methods for detecting and quantifying nitrated lactoferrin (NLF) are lacking. We developed a procedure to selectively quantify NLF using sandwich enzyme-linked immunosorbent assay (ELISA), utilizing a polyclonal anti-LF capture antibody paired with a monoclonal anti-nitrotyrosine detector antibody. The assay was applied to quantify NLF in samples of pure LF nitrated via two separate reactions at molar ratios of excess nitrating agent to the total number of tyrosine residues between 10/1 and 100/1. Tetranitromethane (TNM) was used as a laboratory surrogate for an environmental pathway selective for production of 3-nitrotyrosine, and sodium peroxynitrite (ONOO-) was used as a surrogate for an endogenous nitration pathway. UV-vis spectroscopy (increased absorbance at 350nm) and fluorescence spectroscopy (emission decreased by > 96%) for each reaction indicate the production of NLF. A lower limit of NLF detection using the ELISA method introduced here was calculated to be 0.065μgmL-1, which will enable the detection of human-physiologically relevant concentrations of NLF. Our approach provides a relatively inexpensive and practical way to assess NLF in a variety of systems. Graphical abstract We developed a procedure to selectively quantify nitrated lactoferrin (NLF) protein using a sandwich enzyme-linked immunosorbent assay (ELISA) and verified results against several spectroscopic techniques. Our approach provides an inexpensive and practical way to assess NLF in a variety of systems.

Potential allelopathic effect of Brachiaria decumbens root exudates on neotropical tree seedlings

The reforestation of degraded areas in the neotropics is hampered by the invasion of Brachiaria spp. The aggressiveness of these grasses may be related to the release of allelochemicals in the soil, and the sensitivity of tree seedlings of distinct successional classes to exudates of Brachiaria roots has not been addressed. Here, we evaluated the effects of exudates from Brachiaria decumbens roots on the growth, physiology and nitrogen metabolism of seedlings of six tree species native to Brazilian Atlantic Forest: Trema micrantha, Heliocarpus popayanensis and Cecropia pachystachya (pioneers), Cariniana estrellensis, Eugenia brasiliensis and Guarea kunthiana (non-pioneers). The seedlings were cultivated in fertile soil without (control) and with B. decumbens root exudates (AL treatment). Total leaf area and root and shoot dry weights of seedlings of pioneer species were drastically reduced by AL, whereas non-pioneers were less negatively affected. The gas exchange parameters and chlorophyll content of seedlings of both successional groups were altered by AL. AL decreased endogenous nitrate contents in the seedlings, in accordance with the inhibition of soil nitrification by this treatment. As consequence, AL reduced the endogenous protein contents of pioneer seedlings. Overall, these results demonstrate the negative allelopathic potential of exudates from B. decumbens roots on neotropical tree seedlings. Pioneer species, which are highly dependent on nitrate, were more negatively affected by the grass exudates, suggesting an indirect allelopathic effect mediated by the inhibition of soil nitrification, which may hamper the restoration of degraded areas. However, direct allelopathic mechanisms cannot be ruled out.

The Untapped Potential of Nitroproteomics for Medicine

The endogenous low-abundance tyrosine nitration in a proteome has sensitive biological effect and is a stable biomarker of oxidative/nitrosative stress or injuries, which is closely associated with a wide spectrum of diseases. Some nitroproteomics approaches have been established to achieve the identity of nitoproteins and nitrotyrosine sites; however, high-throughput quantitative nitroproteomics is urgently needed to maximize the coverage of tyrosine nitration in a proteome. The hot research topics would be the competition of nitration and phosphorylation at the same tyrosine residue, and tyrosine nitration within important protein domains and motifs, with pathway network analysis, X-ray crystallography analysis, and fixed point mutation methods to reveal scientific merit of protein nitration in clarification of molecular mechanism of a disease and discovery of effective therapeutic targets. The tyrosine nitration pattern of biofluid might benefit the discovery of reliable biomarkers for accurately predictive, diagnosis, and prognosis assessment of a disease.

451 - Endogenous vs. Dietary Nitrate and Nitrite Distribution in Rat Skeletal Muscle during Exercise

Nitric oxide (NO), small gaseous molecule generated enzymatically by a family of NOS enzymes, is a potent vasodilator. At low oxygen conditions, NO is generated in a series of nonenzymatic reactions from nitrite and nitrate. In the past decade, these two ions started to be considered as storage and precursors molecules for NO. We recently showed that skeletal muscle is the largest body reservoir of nitrate with levels 2-3 times higher than those found in blood or any other internal organ. We proposed that this endogenous nitrate serves as a precursor for NO in muscle during the exercise-induced hypoxia and following active hyperemia. It is known that dietary nitrate intake increases exercise endurance, even if the exact mechanism is still up to debate. In our study, using the combination of dietary nitrate manipulation and exercise, we try to understand relative roles of skeletal muscle endogenous nitrate and exogenous (dietary) nitrate at resting state and during exercise. We showed that the endogenous nitrate stored in skeletal muscle is extensively used by the whole body at times of low nitrate intake in both, resting and exercising state. Dietary nitrate excess is delivered by bloodstream mainly into liver (and likely into other internal organs), but only in very low extent into muscle tissue. However, when on excess of nitrate, nitrite drastically increased in all studied organs. We hypothesize that it is the nitrite, formed in liver from dietary nitrate excess that is transported into skeletal muscle during exercise where it improves performance. The mechanism by which exercise enhances the transport of nitrite derived from dietary sources into skeletal muscle is still unclear.

Rapid induction of GFP expression by the nitrate reductase promoter in the diatom Phaeodactylum tricornutum.

An essential prerequisite for a controlled transgene expression is the choice of a suitable promoter. In the model diatom Phaeodactylum tricornutum, the most commonly used promoters for trans-gene expression are the light dependent lhcf1 promoters (derived from two endogenous genes encoding fucoxanthin chlorophyll a/c binding proteins) and the nitrate dependent nr promoter (derived from the endogenous nitrate reductase gene). In this study, we investigated the time dependent expression of the green fluorescent protein (GFP) reporter under control of the nitrate reductase promoter in independently genetically transformed P. tricornutum cell lines following induction of expression by change of the nitrogen source in the medium via flow cytometry, microscopy and western blotting. In all investigated cell lines, GFP fluorescence started to increase 1 h after change of the medium, the fastest increase rates were observed between 2 and 3 h. Fluorescence continued to increase slightly for up to 7 h even after transfer of the cells to ammonium medium. The subsequent decrease of GFP fluorescence was much slower than the increase, probably due to the stability of GFP. The investigation of several cell lines transformed with nr based constructs revealed that, also in the absence of nitrate, the promoter may show residual activity. Furthermore, we observed a strong variation of gene expression between independent cell lines, emphasising the importance of a thorough characterisation of genetically modified cell lines and their individual expression patterns.

Physiological recycling of endogenous nitrate by oral bacteria regulates gastric mucus thickness

BackgroundInorganic nitrate from exogenous and endogenous sources is accumulated in saliva, reduced to nitrite by oral bacteria and further converted to nitric oxide (NO) and other bioactive nitrogen oxides in the acidic gastric lumen. To further explore the role of oral microbiota in this process we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite. MethodsMice were given either nitrate (100mg/kg/d) or nitrite (0.55–11mg/kg/d) in the drinking water for 7 days, with the lowest nitrite dose resembling the levels provided by swallowing of fasting saliva. The gastric mucus layer was measured in vivo. ResultsGF animals were almost devoid of the firmly adherent mucus layer compared to conventional mice. Dietary nitrate increased the mucus thickness in conventional animals but had no effect in GF mice. In contrast, nitrite at all doses, restored the mucus thickness in GF mice to the same levels as in conventional animals. The nitrite-mediated increase in gastric mucus thickness was not inhibited by the soluble guanylyl cyclase inhibitor ODQ. Mice treated with antibiotics had significantly thinner mucus than controls. Additional studies on mucin gene expression demonstrated down regulation of Muc5ac and Muc6 in germ free mice after nitrite treatment. ConclusionOral bacteria remotely modulate gastric mucus generation via bioactivation of salivary nitrate. In the absence of a dietary nitrate intake, salivary nitrate originates mainly from NO synthase. Thus, oxidized NO from the endothelium and elsewhere is recycled to regulate gastric mucus homeostasis.

Skeletal muscle as an endogenous nitrate reservoir

The nitric oxide synthase (NOS) family of enzymes form nitric oxide (NO) from arginine in the presence of oxygen. At reduced oxygen availability NO is also generated from nitrate in a two step process by bacterial and mammalian molybdopterin proteins, and also directly from nitrite by a variety of five-coordinated ferrous hemoproteins. The mammalian NO cycle also involves direct oxidation of NO to nitrite, and both NO and nitrite to nitrate by oxy-ferrous hemoproteins. The liver and blood are considered the sites of active mammalian NO metabolism and nitrite and nitrate concentrations in the liver and blood of several mammalian species, including human, have been determined. However, the large tissue mass of skeletal muscle had not been generally considered in the analysis of the NO cycle, in spite of its long-known presence of significant levels of active neuronal NOS (nNOS or NOS1). We hypothesized that skeletal muscle participates in the NO cycle and, due to its NO oxidizing heme protein, oxymyoglobin has high concentrations of nitrate ions. We measured nitrite and nitrate concentrations in rat and mouse leg skeletal muscle and found unusually high concentrations of nitrate but similar levels of nitrite, when compared to the liver. The nitrate reservoir in muscle is easily accessible via the bloodstream and therefore nitrate is available for transport to internal organs where it can be reduced to nitrite and NO. Nitrate levels in skeletal muscle and blood in nNOS−/− mice were dramatically lower when compared with controls, which support further our hypothesis. Although the nitrate reductase activity of xanthine oxidoreductase in muscle is less than that of liver, the residual activity in muscle could be very important in view of its total mass and the high basal level of nitrate. We suggest that skeletal muscle participates in overall NO metabolism, serving as a nitrate reservoir, for direct formation of nitrite and NO, and for determining levels of nitrate in other organs.

13 - Skeletal Muscle as an Endogenous Nitrate Reservoir

13 - Skeletal Muscle as an Endogenous Nitrate Reservoir