Showing papers on "Nitrite published in 2005"

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Abstract: Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.

The emerging biology of the nitrite anion

Abstract: Nitrite has now been proposed to play an important physiological role in signaling, blood flow regulation and hypoxic nitric oxide homeostasis. A recent two-day symposium at the US National Institutes of Health highlighted recent advances in the understanding of nitrite biochemistry, physiology and therapeutics.

Chemical conversion of nitrate and nitrite to nitrous oxide for nitrogen and oxygen isotopic analysis in freshwater and seawater.

Abstract: We present a novel method for nitrogen and oxygen natural isotopic abundance analysis of nitrate and nitrite of seawater and freshwater at environmental concentrations. The method involves the reduction of nitrate to nitrite using spongy cadmium with further reduction to nitrous oxide using sodium azide in an acetic acid buffer. For separate nitrite analysis, the cadmium reduction step is simply bypassed. Nitrous oxide is purged from the water sample and trapped cryogenically using an automated system with subsequent release into a gas chromatography column. The isolated nitrous oxide is then analyzed on a continuous flow isotope ratio mass spectrometer via an open split. This paper describes the basic protocol and reaction conditions required to obtain reproducible natural abundance level nitrogen and oxygen isotopic ratios from nitrate, nitrite, or both, and the results obtained to support these conclusions. A standard deviation less than 0.2‰ for nitrogen and 0.5‰ for oxygen was found for nitrate sampl...

Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control.

Abstract: Hypoxic vasodilation is a fundamental, highly conserved physiological response that requires oxygen and/or pH sensing coupled to vasodilation. While this process was first characterized more than 80 years ago, the precise identity and mechanism of the oxygen sensor and mediators of vasodilation remain uncertain. In support of a possible role for hemoglobin (Hb) as a sensor and effector of hypoxic vasodilation, here we show biochemical evidence that Hb exhibits enzymatic behavior as a nitrite reductase, with maximal NO generation rates occurring near the oxy-to-deoxy (R-to-T) allosteric structural transition of the protein. The observed rate of nitrite reduction by Hb deviates from second-order kinetics, and sigmoidal reaction progress is determined by a balance between 2 opposing chemistries of the heme in the R (oxygenated conformation) and T (deoxygenated conformation) allosteric quaternary structures of the Hb tetramer — the greater reductive potential of deoxyheme in the R state tetramer and the number of unligated deoxyheme sites necessary for nitrite binding, which are more plentiful in the T state tetramer. These opposing chemistries result in a maximal nitrite reduction rate when Hb is 40–60% saturated with oxygen (near the Hb P50), an apparent ideal set point for hypoxia-responsive NO generation. These data suggest that the oxygen sensor for hypoxic vasodilation is determined by Hb oxygen saturation and quaternary structure and that the nitrite reductase activity of Hb generates NO gas under allosteric and pH control.

Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues

Abstract: Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO2(-)). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo. We further find that nitrite readily affects cyclic GMP production, cytochrome P450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo, suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.

Nitric oxide emission from tobacco leaves and cell suspensions: rate limiting factors and evidence for the involvement of mitochondrial electron transport.

Abstract: Quantitative data on nitric oxide (NO) production by plants, and knowledge of participating reactions and rate limiting factors are still rare. We quantified NO emission from tobacco (Nicotiana tabacum) wild-type leaves, from nitrate reductase (NR)- or nitrite reductase (NiR)-deficient leaves, from WT- or from NR-deficient cell suspensions and from mitochondria purified from leaves or cells, by following NO emission through chemiluminescence detection. In all systems, NO emission was exclusively due to the reduction of nitrite to NO, and the nitrite concentration was an important rate limiting factor. Using inhibitors and purified mitochondria, mitochondrial electron transport was identified as a major source for reduction of nitrite to NO, in addition to NR. NiR and xanthine dehydrogenase appeared to be not involved. At equal respiratory activity, mitochondria from suspension cells had a much higher capacity to produce NO than leaf mitochondria. NO emission in vivo by NiR-mutant leaves (which was not nitrite limited) was proportional to photosynthesis (high in light +CO(2), low in light -CO(2), or in the dark). With most systems including mitochondrial preparations, NO emission was low in air (and darkness for leaves), but high under anoxia (nitrogen). In contrast, NO emission by purified NR was not much different in air and nitrogen. The low aerobic NO emission of darkened leaves and cell suspensions was not due to low cytosolic NADH, and appeared only partly affected by oxygen-dependent NO scavenging. The relative contribution of NR and mitochondria to nitrite-dependent NO production is estimated.

NO Generation From Nitrite and Its Role in Vascular Control

Abstract: NO generated from L-arginine by NO synthases (NOSs) in the endothelium and in other cells plays a central role in several aspects of vascular biology. The biological activity of NO is acutely terminated by oxidation to nitrite and nitrate, and these compounds have long been considered only as inert end-products of NO. However, this dogma is now being challenged because recent research convincingly has shown that the nitrite ion can be recycled back to bioactive NO again in blood and tissues. Nitrite reduction to NO can occur via several routes involving enzymes, proteins, vitamins, or even simple protons. This pathway may serve as a backup system for NO generation in conditions such as hypoxia, in which the NOS/L-arginine system is compromised, but detrimental effects can also be foreseen. With this new knowledge, nitrate and nitrite should probably be viewed as storage pools for NO rather than inert waste products. Here we discuss novel aspects of nitrite-dependent NO generation in vivo and its role in vascular control.

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids.

Abstract: In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from l-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects consitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most si...

Nitric oxide signaling in plants

Abstract: Plants have four nitric oxide synthase (NOS) enzymes. NOS1 appears mitochondrial, and inducible nitric oxide synthase (iNOS) chloroplastic. Distinct peroxisomal and apoplastic NOS enzymes are predicted. Nitrite-dependent NO synthesis is catalyzed by cytoplasmic nitrate reductase or a root plasma membrane enzyme, or occurs nonenzymatically. Nitric oxide undergoes both catalyzed and uncatalyzed oxidation. However, there is no evidence of reaction with superoxide, and S-nitrosylation reactions are unlikely except during hypoxia. The only proven direct targets of NO in plants are metalloenzymes and one metal complex. Nitric oxide inhibits apoplastic catalases/ascorbate peroxidases in some species but may stimulate these enzymes in others. Plants also have the NO response pathway involving cGMP, cADPR, and release of calcium from internal stores. Other known targets include chloroplast and mitochondrial electron transport. Nitric oxide suppresses Fenton chemistry by interacting with ferryl ion, preventing generation of hydroxyl radicals. Functions of NO in plant development, response to biotic and abiotic stressors, iron homeostasis, and regulation of respiration and photosynthesis may all be ascribed to interaction with one of these targets. Nitric oxide function in drought/abscisic acid (ABA)-induction of stomatal closure requires nitrate reductase and NOS1. Nitric oxide synthasel likely functions to produce sufficient NO to inhibit photosynthetic electron transport, allowing nitrite accumulation. Nitric oxide is produced during the hypersensitive response outside cells undergoing programmed cell death immediately prior to loss of plasma membrane integrity. A plasma membrane lipid-derived signal likely activates apoplastic NOS. Nitric oxide diffuses within the apoplast and signals neighboring cells via hydrogen peroxide (H2O2)-dependent induction of salicylic acid biosynthesis. Response to wounding appears to involve the same NOS and direct targets.

In higher plants, only root mitochondria, but not leaf mitochondria reduce nitrite to NO, in vitro and in situ.

Abstract: At oxygen concentrations of < or =1%, even completely nitrate reductase (NR)-free root tissues reduced added nitrite to NO, indicating that, in roots, NR was not the only source for nitrite-dependent NO formation. By contrast, NR-free leaf slices were not able to reduce nitrite to NO. Root NO formation was blocked by inhibitors of mitochondrial electron transport (Myxothiazol and SHAM), whereas NO formation by NR-containing leaf slices was insensitive to the inhibitors. Consistent with that, mitochondria purified from roots, but not those from leaves, reduced nitrite to NO at the expense of NADH. The inhibitor studies suggest that, in root mitochondria, both terminal oxidases participate in NO formation, and they also suggest that even in NR-containing roots, a large part of the reduction of nitrite to NO was catalysed by mitochondria, and less by NR. The differential capacity of root and leaf mitochondria to reduce nitrite to NO appears to be common among higher plants, since it has been observed with Arabidopsis, barley, pea, and tobacco. A specific role for nitrite to NO reduction in roots under anoxia is discussed.

Ammonium oxidation coupled to dissimilatory reduction of iron under anaerobic conditions in wetland soils

Abstract: In exploring the dynamics of iron and nitrogen cycling in sediments from riparian forests we have observed a redox reaction that has not been previously described. During incubations of soil slurries under strictly anaerobic conditions, we repeatedly measured an unexpected production of both nitrite ( NO 2 − ) and ferrous iron [Fe(II)]. Using this indirect evidence we hypothesize that, under anaerobic conditions, there is a biological process that uses ferric iron [Fe(III)] as an electron acceptor while oxidizing ammonium ( NH 4 + ) to NO 2 − for energy production. This NH 4 + oxidation under iron reducing anaerobic conditions is thermodynamically feasible and is potentially a critical component of the N cycle in saturated sediments.

Nitrite infusions to prevent delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage

Abstract: ContextDelayed cerebral vasospasm causes permanent neurological deficits or death in at least 15% of patients following otherwise successful treatment for ruptured intracranial aneurysm. Decreased bioavailability of nitric oxide has been associated with the development of cerebral vasospasm.ObjectiveTo determine whether infusions of nitrite will prevent delayed cerebral vasospasm.Design, Setting, and SubjectsA total of 14 anesthetized cynomolgus monkeys had an autologous blood clot placed around the right middle cerebral artery. Cerebral arteriography was performed before clot placement and on days 7 and 14 to assess vasospasm. The study was conducted from August 2003 to February 2004.InterventionsA 90-mg sodium nitrite intravenous solution infused over 24 hours plus a 45-mg sodium nitrite bolus daily (n = 3); a 180-mg sodium nitrite intravenous solution infused over 24 hours (n = 3); or a control saline solution infusion (n = 8). Each was infused continuously for 14 days.Main Outcome MeasuresNitrite, S-nitrosothiol, and methemoglobin levels in blood and cerebrospinal fluid and degree of arteriographic vasospasm.ResultsIn control monkeys, mean (SD) cerebrospinal fluid nitrite levels decreased from 3.1 (1.5) μmol/L to 0.4 (0.1) μmol/L at day 7 and to 0.4 (0.4) μmol/L at day 14 (P = .03). All 8 control monkeys developed significant vasospasm of the right middle cerebral artery, which was complicated by stroke and death in 1 animal. Sodium nitrite infusions increased the nitrite and methemoglobin levels (<2.1% of total hemoglobin) in the blood and cerebrospinal fluid without evoking systemic hypotension. Nitrite infusion prevented development of vasospasm (no animals developed significant vasospasm; mean [SD] reduction in right middle cerebral artery area on day 7 after subarachnoid hemorrhage of 8% [9%] in nitrite-treated monkeys vs 47% [5%] in saline-treated controls; P<.001). There was a negative correlation between the concentration of nitrite in cerebrospinal fluid and the degree of cerebral vasospasm (P<.001). Pharmacological effects of nitrite infusion were also associated with the formation of S-nitrosothiol in cerebrospinal fluid. There was no clinical or pathological evidence of nitrite toxicity.ConclusionSubacute sodium nitrite infusions prevented delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.

Photosynthetic nitrate assimilation in cyanobacteria.

Abstract: Nitrate uptake and reduction to nitrite and ammonium are driven in cyanobacteria by photosynthetically generated assimilatory power, i.e., ATP and reduced ferredoxin. High-affinity nitrate and nitrite uptake takes place in different cyanobacteria through either an ABC-type transporter or a permease from the major facilitator superfamily (MFS). Nitrate reductase and nitrite reductase are ferredoxin-dependent metalloenzymes that carry as prosthetic groups a [4Fe-4S] center and Mo-bis-molybdopterin guanine dinucleotide (nitrate reductase) and [4Fe-4S] and siroheme centers (nitrite reductase). Nitrate assimilation genes are commonly found forming an operon with the structure: nir (nitrite reductase)-permease gene(s)-narB (nitrate reductase). When the cells perceive a high C to N ratio, this operon is transcribed from a complex promoter that includes binding sites for NtcA, a global nitrogen-control regulator that belongs to the CAP family of bacterial transcription factors, and NtcB, a pathway-specific regulator that belongs to the LysR family of bacterial transcription factors. Transcription is also affected by other factors such as CnaT, a putative glycosyl transferase, and the signal transduction protein P(II). The latter is also a key factor for regulation of the activity of the ABC-type nitrate/nitrite transporter, which is inhibited when the cells are incubated in the presence of ammonium or in the absence of CO(2). Notwithstanding significant advance in understanding the regulation of nitrate assimilation in cyanobacteria, further post-transcriptional regulatory mechanisms are likely to be discovered.

Correlation between anammox activity and microscale distribution of nitrite in a subtropical mangrove sediment

Abstract: The distribution of anaerobic ammonium oxidation (anammox) in nature has been addressed by only a few environmental studies, and our understanding of how anammox bacteria compete for substrates in natural environments is therefore limited. In this study, we measure the potential anammox rates in sediment from four locations in a subtropical tidal river system. Porewater profiles of NOx− (NO2− plus NO3−) and NO2− were measured with microscale biosensors, and the availability of NO2− was compared with the potential for anammox activity. The potential rate of anammox increased with increasing distance from the mouth of the river and correlated strongly with the production of nitrite in the sediment and with the average concentration or total pool of nitrite in the suboxic sediment layer. Nitrite accumulated both from nitrification and from NOx− reduction, though NOx− reduction was shown to have the greatest impact on the availability of nitrite in the suboxic sediment layer. This finding suggests that denitrification, though using NO2− as a substrate, also provides a substrate for the anammox process, which has been suggested in previous studies where microscale NO2− profiles were not measured.

Identification of Bacteria in Biofilm and Bulk Water Samples from a Nonchlorinated Model Drinking Water Distribution System: Detection of a Large Nitrite-Oxidizing Population Associated with Nitrospira spp.

Abstract: In a model drinking water distribution system characterized by a low assimilable organic carbon content (<10 microg/liter) and no disinfection, the bacterial community was identified by a phylogenetic analysis of rRNA genes amplified from directly extracted DNA and colonies formed on R2A plates. Biofilms of defined periods of age (14 days to 3 years) and bulk water samples were investigated. Culturable bacteria were associated with Proteobacteria and Bacteriodetes, whereas independently of cultivation, bacteria from 12 phyla were detected in this system. These included Acidobacteria, Nitrospirae, Planctomycetes, and Verrucomicrobia, some of which have never been identified in drinking water previously. A cluster analysis of the population profiles from the individual samples divided biofilms and bulk water samples into separate clusters (P = 0.027). Bacteria associated with Nitrospira moscoviensis were found in all samples and encompassed 39% of the sequenced clones in the bulk water and 25% of the biofilm community. The close association with Nitrospira suggested that a large part of the population had an autotrophic metabolism using nitrite as an electron donor. To test this hypothesis, nitrite was added to biofilm and bulk water samples, and the utilization was monitored during 15 days. A first-order decrease in nitrite concentration was observed for all samples with a rate corresponding to 0.5 x 10(5) to 2 x 10(5) nitrifying cells/ml in the bulk water and 3 x 10(5) cells/cm(2) on the pipe surface. The finding of an abundant nitrite-oxidizing microbial population suggests that nitrite is an important substrate in this system, potentially as a result of the low assimilable organic carbon concentration. This finding implies that microbial communities in water distribution systems may control against elevated nitrite concentrations but also contain large indigenous populations that are capable of assisting the depletion of disinfection agents like chloramines.

The complete denitrification pathway of the symbiotic, nitrogen-fixing bacterium Bradyrhizobium japonicum.

Abstract: Denitrification is an alternative form of respiration in which bacteria sequentially reduce nitrate or nitrite to nitrogen gas by the intermediates nitric oxide and nitrous oxide when oxygen concentrations are limiting. In Bradyrhizobium japonicum , the N2-fixing microsymbiont of soya beans, denitrification depends on the napEDABC , nirK , norCBQD , and nosRZDFYLX gene clusters encoding nitrate-, nitrite-, nitric oxide- and nitrous oxide-reductase respectively. Mutational analysis of the B. japonicum nap genes has demonstrated that the periplasmic nitrate reductase is the only enzyme responsible for nitrate respiration in this bacterium. Regulatory studies using transcriptional lacZ fusions to the nirK , norCBQD and nosRZDFYLX promoter region indicated that microaerobic induction of these promoters is dependent on the fixLJ and fixK 2 genes whose products form the FixLJ–FixK2 regulatory cascade. Besides FixK2, another protein, nitrite and nitric oxide respiratory regulator, has been shown to be required for N-oxide regulation of the B. japonicum nirK and norCBQD genes. Thus nitrite and nitric oxide respiratory regulator adds to the FixLJ–FixK2 cascade an additional control level which integrates the N-oxide signal that is critical for maximal induction of the B. japonicum denitrification genes. However, the identity of the signalling molecule and the sensing mechanism remains unknown. Abbreviations: CRP, cAMP receptor protein; Fnr, fumarate and nitrate reductase; Nap, periplasmic nitrate reductase; Nar, membrane-bound nitrate reductase; Nir, nitrite reductase; NnrR, nitrite and nitric oxide respiratory regulator; Nor, nitric oxide reductase; Nos, nitrous oxide reductase; Tat, twin arginine translocon

Respirometric estimation of the oxygen affinity constants for biological ammonium and nitrite oxidation

Abstract: The nitrification process (ie biological ammonium oxidation to nitrate) is a two-step process with nitrite as an intermediate product. As it is an aerobic process, its kinetics is highly dependent on the dissolved oxygen (DO) concentration in the medium. However, the influence of this limitation on the nitritation (first step) is shown to be less important than in the nitratation (second step). This dependence on DO concentration is generally described using a Monod-type kinetics with KO as the oxygen affinity constant. In this work, a procedure for the calculation of both affinity constants is presented. This procedure is based on monitoring the DO drop in the reactor when external aeration is stopped and the biomass is consuming without substrate (ammonium or nitrite) limitations. This methodology includes the contemplation of the oxygen transfer from the atmosphere, the response time of the DO probe and the inhibition of the nitratation step with sodium azide when estimating KOA (nitritation oxygen affinity constant). The results obtained are KOA = 0.74 ± 0.02 mg O2 dm−3 and KON = 1.75 ± 0.01 mg O2 dm−3. Moreover the influence of the aforementioned considerations on the estimated KO values is also discussed. Copyright © 2005 Society of Chemical Industry

Atomic Resolution Structures of Resting-State, Substrate- and Product-Complexed Cu-Nitrite Reductase Provide Insight Into Catalytic Mechanism

Abstract: Copper-containing nitrite reductases catalyze the reduction of nitrite to nitric oxide (NO), a key step in denitrification that results in the loss of terrestrial nitrogen to the atmosphere. They are found in a wide variety of denitrifying bacteria and fungi of different physiology from a range of soil and aquatic ecosystems. Structural analysis of potential intermediates in the catalytic cycle is an important goal in understanding enzyme mechanism. Using "crystal harvesting" and substrate-soaking techniques, we have determined atomic resolution structures of four forms of the green Cu-nitrite reductase, from the soil bacterium Achromobacter cycloclastes. These structures are the resting state of the enzyme at 0.9 A, two species exhibiting different conformations of nitrite bound at the catalytic type 2 Cu, one of which is stable and also has NO present, at 1.10 A and 1.15 A, and a stable form with the product NO bound side-on to the catalytic type 2 Cu, at 1.12 A resolution. These structures provide incisive insights into the initial binding of substrate, its repositioning before catalysis, bond breakage (O-NO), and the formation of a stable NO adduct.