Intracellular Nitrite Research Articles

Regulation of nitrite transport in red blood cells by hemoglobin oxygen fractional saturation

Allosteric regulation of nitrite reduction by deoxyhemoglobin has been proposed to mediate nitric oxide (NO) formation during hypoxia. Nitrite is predominantly an anion at physiological pH, raising questions about the mechanism by which it enters the red blood cell (RBC) and whether this is regulated and coupled to deoxyhemoglobin-mediated reduction. We tested the hypothesis that nitrite transport by RBCs is regulated by fractional saturation. Using human RBCs, nitrite consumption was faster at lower fractional saturations, consistent with faster reactions with deoxyheme. A membrane-based regulation was suggested by slower nitrite consumption with intact versus lysed RBCs. Interestingly, upon nitrite addition, intracellular nitrite concentrations attained a steady state that, despite increased rates of consumption, did not change with decreasing oxygen tensions, suggesting a deoxygenation-sensitive step that either increases nitrite import or decreases the rate of nitrite export. A role for anion exchanger (AE)-1 in the control of nitrite export was suggested by increased intracellular nitrite concentrations in RBCs treated with DIDS. Moreover, deoxygenation decreased steady-state levels of intracellular nitrite in AE-1-inhibited RBCs. Based on these data, we propose a model in which deoxyhemoglobin binding to AE-1 inhibits nitrite export under low oxygen tensions allowing for the coupling between deoxygenation and nitrite reduction to NO along the arterial-to-venous gradient.

Effects of nitrate on intracellular nitrite and growth of Microcystis aeruginosa

Although nitrate is a macronutrient and can serve as good nitrogen source for many species of phytoplankton, high nitrate concentrations do not benefit the growth of phytoplankton. We hypothesise that algae cultured under high nitrate concentrations can accumulate intracellular nitrite, which is produced by nitrate reductase (NR) and can inhibit the growth of algae. To assess the validity of this hypothesis, Microcystis aeruginosa was grown under different nitrate concentrations from 3.57 to 21.43 mM in low CO2 and high CO2 conditions for 15 days. We observed that, with increasing nitrate concentrations, the intracellular nitrite concentrations of the alga increased and the growth rates and photosynthesis declined. When grown under high CO2 conditions, M. aeruginosa showed lower intracellular nitrite concentrations and higher growth rates and $${\text{P}}_{\text{m}}^{{\text{chl}}a} $$ , $${\text{R}}_{\text{d}}^{{\text{chl}}a} $$ , αchla than under low CO2 conditions. These results suggest that the accumulation of intracellular nitrite could be the cause of inhibition of algal growth under high nitrate concentrations.

Oxidation of Iron-nitrosyl-hemoglobin by Dehydroascorbic Acid Releases Nitric Oxide to Form Nitrite in Human Erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.

Nitric oxide synthase reduces nitrite to NO under anoxia.

Cultured bEND.3 endothelial cells show a marked increase in NO production when subjected to anoxia, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The rate of anoxic NO production exceeds basal unstimulated NO synthesis in normoxic cells. The anoxic release of NO is mediated by endothelial nitric oxide synthase (eNOS), can be abolished by inhibitors of NOS and is accompanied by consumption of intracellular nitrite. The anoxic NO release is unaffected by the xanthine oxidase inhibitor oxypurinol. The phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS, and its magnitude and duration suggests that the nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues.

Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of all aquatic food chains by fixing carbon and nitrogen into cellular biomass. The single most important nutrient for photosynthesis and growth is nitrate, which is severely limiting in many aquatic environments particularly the open ocean. It is therefore not surprising that NrtA, the solute-binding component of the high-affinity nitrate ABC transporter, is the single-most abundant protein in the plasma membrane of these bacteria. Here, we describe the structure of a nitrate-specific receptor, NrtA from Synechocystis sp. PCC 6803, complexed with nitrate and determined to a resolution of 1.5 A. NrtA is significantly larger than other oxyanion-binding proteins, representing a previously uncharacterized class of transport proteins. From sequence alignments, the only other solute-binding protein in this class is CmpA, a bicarbonate-binding protein. Therefore, these organisms created a solute-binding protein for two of the most important nutrients: inorganic nitrogen and carbon. The electrostatic charge distribution of NrtA appears to force the protein off the membrane while the flexible tether facilitates the delivery of nitrate to the membrane pore. The structure not only details the determinants for nitrate selectivity in NrtA but also the bicarbonate specificity in CmpA. Nitrate and bicarbonate transport are regulated by the cytoplasmic proteins NrtC and CmpC, respectively. Interestingly, the residues lining the ligand binding pockets suggest that they both bind nitrate. This implies that the nitrogen and carbon uptake pathways are synchronized by intracellular nitrate and nitrite.

Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus.

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.

Nitrate Reductase Alters 3-Nitrotyrosine Accumulation and Cell Cycle Progression in LPS + IFN-γ-Stimulated RAW 264.7 Cells

Nitrite (NO−2), an end product of nitrogen radical metabolism, has recently been shown to increase tyrosine nitration by activated leukocytes indicating that nitrite modulates the immune response. We investigated the hypothesis that nitrite may increase nitration of molecular targets within activated cells leading to altered cell cycle progression. Intracellular nitrite was increased by transfection of murine macrophage-like RAW 264.7 cells with the nitrate reductase gene obtained from barley. Nitrate reductase facilitates the conversion of nitrate to nitrite; thus when extracellular nitrate is present, intracellular nitrite will be increased. Results show that addition of KNO3increases NO−2production and intracellular nitrotyrosine accumulation in the transfectant but not the parent. Inhibition of nitric oxide synthesis with L-NAME during activation with IFN-γ + LPS reduced NO−2production to the same extent in both cell lines; however, cellular accumulation of nitrotyrosine was reduced by only 25% in the transfectant (P= 0.21) and 49% in the parent cell line (P= 0.007), suggesting that intracellular nitrite increased nitrotyrosine accumulation through a pathway not requiring NO synthesis, i.e., myeloperoxidase system. Approximately 15% of the transfected cells had 4n DNA content 24 h postactivation compared to <1% of the parent cells. Increased DNA copy number was correlated to nitrotyrosine accumulation. These findings show that intracellular nitrite can increase accumulation of nitrotyrosine and that nitration is linked to cell cycle perturbation.

Recovery of nitrogen-starved cultures of the diatom Ditylum brightwellii (Bacillariophyceae) upon nitrogen resupply

N-limited continuous cultures of the diatom Ditylum brightwellii were starved for nitrogen to study the effects of nitrogen pulses on algal growth and cell lysis kinetics. Growth rate responses after N-replenishment did not differ for the three types of nitrogen (nitrate, ammonium and nitrite) added, but death rate responses did. Resupply of 200 μM nitrate to cultures preconditioned at a steady-state growth rate (μ) of 0.53 d −1 resulted in enhanced algal cell lysis, with average maximum specific cell lysis rates ( d b) of 0.24±0.07 d −1 compared to d b<0.06 d −1 for the control populations. Enhanced cell lysis was also found upon addition with nitrite, but not with ammonium. Algal mortality rates reduced with decreasing concentrations of nitrate additions, and with decreasing starvation time that the cultures were subjected to. Populations of D. brightwellii precultured at 0.30 d −1 did not show accelerated death responses to nitrate addition. The pH in the cultures was generally >8.5. Cultures preconditioned at 0.53 d −1 and pulsed with nitrate gave no enhanced d b when buffered at pH 8±0.1 with 20 mM HEPES. A light/dark cycle appeared essential to obtain accelerated algal cell death. Increased irradiance enhanced cell lysis strongly, whereas subsaturating irradiance levels reduced algal mortality. Accumulation of nitrite intracellularly indicated that the reduction of nitrite to ammonium was the rate-limiting step in nitrate assimilation. It is argued that the hypothetical mechanism behind the enhanced death responses concerns excretion of intracellular nitrite by active transport and deregulation of the protonmotive across the algal cell membranes, but this remains to be studied.

Determination of Intracellular and Extracellular Nitrite and Nitrate by Anion Chromatography

Abstract A highly sensitive method for the simultaneous direct detection as well as the ratio of intracellular and extracellular nitrite (NO2 −) and nitrate (NO3 −) in human macrophage (Mφ), plasma and urine has been developed. Samples were deproteinized with the organic solvent acetonirile, lyophilized and reconstituted in buffer, determination and quantification of nitric oxide (NO) stable end products NO2 − and NO3 − was performed utilizing an isocratic high performance liquid chromatography (HPLC) with an anion exchange column. A mobile phase of 20 mM NaCl with 1mM mono sodium phosphate-NaH2PO4 at pH = 7.0 was used. Analyte anions were detected by direct UV-wavelength at 210 nm. Sensitivity in intracellular and extracellular fluids were 0.01 μmol/L for both anions with recovery rates of 99.6–99.4% for NO2 − and NO3 −. This method has successfully been applied to the determination of nitrite and nitrate in plasma and urine of normal human volunteers as well as in cell line U-937 macrophage cytosol/supernatant. The mean concentration of NO3 − in normal plasma (n=22) was 3.1 ± 0.4 μmol/L and NO3 − 10.3 ± 0.3 μmol/L with ratio of NO2 −:NO3 − = 0.3; in normal urine (n=22) NO2 − was 380 ± 61 μmol/L and NO3 − 1345 ± 270 μmol/L with ratio of NO2 −:NO3 − = 0.3; in intracellular human macrophage fluid (n=21) NO2 − was 0.66 ± 0.08 mol/L/106 Mφ, and NO3 − 0.68 ± 0.09 mol/L/106 Mφ with ratio NO2 −:NO3 = 1; and in extracellular human macrophage fluid (n=21) NO2 − was 0.03 ± 0.01 mol/L/106 Mφ and NO3 − 3.03 ± 0.33 mol/L/106 Mφ with ratio NO2 −:NO3 − = 0.01.

The biochemistry and immunology of yeasts that produce true mycelia: Ambrosiozyma, Hormoascus, and Hyphopichia.

Ambrosiozyma, yeasts that produce true mycelia with transverse septa that communicate by a dolipore type septum, are compared with Hyphopichia and Hormoascus. This study includes intracellular oxidases, nitrite and nitrate reductases, guanine plus cytosine content in DNA (GC-content), cytochrome spectra (a+a3, b, c), coenzyme Q, and serological comparison by immunodiffusion and immunoelectrophoresis, with A. monospora and Hormoascus platypodis as references. The results support the division of the genus Ambrosiozyma into 2 groups based upon GC content, vitamin requirements, and serological homologies. Affinities between Hormoascus platypodis and A. philentoma (vitamin requirements, intracellular nitrite and nitrate reductases, β-galactosidase, serological homology, cytochrome spectra, and GC content) leads to the placing of Hormoascus platypodis in synonymy with Ambrosiozyma philentoma. Ambrosiozyma ambrosiae is compared with the other Ambrosiozyma and with the non-fermentative Pichia, and Hansenula silvicola. These comparisons show that A. ambrosiae has more affinity with the other Ambrosiozyma than with Pichia or Hansenula. Hyphopichia burtonii differs from the other species in regard to vitamin requirements, coenzyme Q8, α2 band of cytochrome c and serological homologies.

Contribution � la syst�matique du genre Pichia

The presence of intracellular osidases, nitrite and nitrate reductases, vitamin requirements and GC content in the DNA, have been investigated for in 18 species of Pichia.

Recherche D'enzymes intracellulaires dans le genre schizosaccharomyces. Implications systématiques

In the genus Schizosaccharomyces intracellular osidases and nitrite and nitrate reductases are revealed; particularly all the species possessing invertase, alpha-glucosidase and alpha-galactosidase. These characters underline the homogeneity on the genus. On the basis of osidases, nitrite and nitrate reductases results, 2 groups can be distinguished in this genus.

Nitrite r�ductase des Saccharomyces (groupe Torulaspora) et des Debaryomyces. implications syst�matiques

Intracellular nitrite reductase was found in some species of Saccharomyces (Torulaspora group of Van der Walt 1970) and all Debaryomyces (excepted D. castelli and D. coudertii. The taxonomical relationship between the 2 groupes are discussed.