sodium-nitrite and peroxynitric-acid

We have recently shown that a wide range of different inorganic salts can potentiate antimicrobial photodynamic inactivation (aPDI) and TiO2-mediated antimicrobial photocatalysis. Potentiation has been shown with azide, bromide, thiocyanate, selenocyanate, and most strongly, with iodide. Here we show that sodium nitrite can also potentiate broad-spectrum aPDI killing of Gram-positive MRSA and Gram-negative Escherichia coli bacteria. Literature reports have previously shown that two photosensitizers (PS), methylene blue (MB) and riboflavin, when excited by broad-band light in the presence of nitrite could lead to tyrosine nitration. Addition of up to 100 mM nitrite gave 6 logs of extra killing in the case of Rose Bengal excited by green light against E. coli, and 2 logs of extra killing against MRSA (eradication in both cases). Comparable results were obtained for other PS (TPPS4 + blue light and MB + red light). Some bacterial killing was obtained when bacteria were added after light using a functionalized fullerene (LC15) + nitrite + blue light, and tyrosine ester amide was nitrated using both "in" and "after" modes with all four PS. The mechanism could involve formation of peroxynitrate by a reaction between superoxide radicals and nitrogen dioxide radicals; formation of the latter species was demonstrated by spin trapping with nitromethane.

Topics: Anti-Bacterial Agents; Drug Synergism; Escherichia coli; Fullerenes; Light; Methicillin-Resistant Staphylococcus aureus; Microbial Viability; Models, Molecular; Molecular Conformation; Nitrates; Sodium Nitrite

Endogenous nitric oxide (NO) reacts with superoxide to form peroxynitrite, which is capable of either oxidizing or nitrating various biological substrates. We compared the vasodilatory effect of exogenous peroxynitrite with the effects of decomposed peroxynitrite or sodium nitrite in precontracted aorta isolated from streptozotocin-induced diabetic and age-matched control rats. Peroxynitrite (10 nmol/l to 300 micromol/l) produced a concentration-dependent relaxation in aortic rings with or without endothelium. Relaxation was also observed with a higher concentration of its decomposition product or sodium nitrite, although these relaxations were considerably slower and with reduced sensitivity. Endothelium-containing rings were less sensitive to the vasorelaxant effect of peroxynitrite than the endothelium-denuded rings in control (pD(2) was 5.19 +/- 0.06 in rings with endothelium and 5.86 +/- 0.03 in rings without endothelium, p < 0.01) but not in diabetic aorta (pD(2) was 5.97 +/- 0.05 in rings with endothelium and 6.12 +/- 0.06 in rings without endothelium, p > 0.05). The maximum relaxation to peroxynitrite also increased in diabetics, but did not change by removal of the endothelium either in diabetic or control rings. Diabetes did not alter the relaxations elicited by both decomposed peroxynitrite and sodium nitrite. Peroxynitrite-induced relaxation was not inhibited by diethylenetriaminepentaacetic acid, an inhibitor of hydroxyl radical formation. Pretreatment with peroxynitrite (1 micromol/l, 15 min) significantly suppressed the phenylephrine-induced tone and acetylcholine-stimulated endothelium-dependent relaxation, both effects were more pronounced in diabetic than in control aorta. The increased responsiveness of diabetic vessels to exogenous peroxynitrite seems to be related to depressed basal NO bioavailability and may be considered as a compensatory way against activated contractile mechanisms of diabetic vascular smooth muscle.

Topics: Acetylcholine; Animals; Aorta; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Endothelium, Vascular; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitrates; Phenylephrine; Rats; Rats, Wistar; Sodium Nitrite; Vasoconstrictor Agents; Vasodilator Agents

Reactive nitrogen species, including nitrogen oxides (N(2)O(3) and N(2)O(4)), peroxynitrite (ONOO(-)), and nitryl chloride (NO(2)Cl), have been implicated as causes of inflammation and cancer. We studied reactions of secondary amines with peroxynitrite and found that both N-nitrosamines and N-nitramines were formed. Morpholine was more easily nitrosated by peroxynitrite at alkaline pH than at neutral pH, whereas its nitration by peroxynitrite was optimal at pH 8.5. The yield of nitrosomorpholine in this reaction was 3 times higher than that of nitromorpholine at alkaline pH, whereas 2 times more nitromorpholine than nitrosomorpholine was formed at pH <7.5. For the morpholine-peroxynitrite reaction, nitration was enhanced by low concentrations of bicarbonate, but was inhibited by excess bicarbonate. Nitrosation was inhibited by excess bicarbonate. On this basis, we propose a free radical mechanism, involving one-electron oxidation by peroxynitrite of secondary amines to form amino radicals (R(2)N(*)), which react with nitric oxide ((*)NO) or nitrogen dioxide ((*)NO(2)) to yield nitroso and nitro secondary amines, respectively. Reaction of morpholine with NO(*) and superoxide anion (O(2)(*)(-)), which were concomitantly produced from spermine NONOate and by the xanthine oxidase systems, respectively, also yielded nitromorpholine, but its yield was <1% of that of nitrosomorpholine. NO(*) alone increased the extent of nitrosomorpholine formation in a dose-dependent manner, and concomitant production of O(2)(*)(-) inhibited its formation. Reactions of morpholine with nitrite plus HOCl or nitrite plus H(2)O(2), with or without addition of myeloperoxidase or horseradish peroxidase, also yielded nitration and nitrosation products, in yields that depended on the reactants. Tyrosine was nitrated easily by synthetic peroxynitrite, by NaNO(2) plus H(2)O(2) with myeloperoxidase, and by NaNO(2) plus H(2)O(2) under acidic conditions. Nitrated secondary amines, e.g., N-nitroproline, could be identified as specific markers for endogenous nitration mediated by reactive nitrogen species.

Topics: Aniline Compounds; Hydrogen Peroxide; Hypochlorous Acid; Morpholines; Nitrates; Nitrobenzenes; Nitrosamines; Sodium Nitrite; Superoxides; Tyrosine

The aim of the present study was to explore the mechanisms by which nitric oxide (NO) may inhibit aromatase activity of human granulosa cells. Ovarian granulosa-luteal cells, obtained from patients undergoing in-vitro fertilization (IVF) were cultured in the presence of NO-related substances. After 24 h of culture, aromatase activity of the cells was significantly inhibited by treatment with the NO donors, SNAP or NOC12 at > or =10(-4) M in a dose-dependent manner. Treatment with NO catabolites or a peroxynitrite-releasing compound, SIN1, had no significant influence. Treatment with SNAP at 10(-3) M decreased relative aromatase mRNA values by 72% (P<0.05) and intracellular cyclic AMP concentrations by 53% (P<0.01). However, treatment with H89, an inhibitor of protein kinase A, did not inhibit aromatase activity. Since there were no significant effects of NO catabolites or peroxinitrite, the inhibitory action of NO donors on aromatase must be related to NO release. The action of NO is, in part, attributable to the down-regulation of aromatase gene transcription. Although NO decreased intracellular cAMP values, down-regulation of aromatase gene transcription may not be mediated by protein kinase A-dependent mechanisms.

Topics: Adult; Aromatase; Aromatase Inhibitors; Cells, Cultured; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Female; Gene Expression Regulation; Granulosa Cells; Humans; Isoquinolines; Nitrates; Nitric Oxide; Penicillamine; Protein Kinase C; Sodium Nitrite; Sulfonamides; Tetradecanoylphorbol Acetate; Transcription, Genetic

Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence for in vivo formation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significance in vivo has not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-microl injection). We have applied this method to compare in vitro the ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholinosydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO-) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.

Topics: Chromatography, High Pressure Liquid; Electrochemistry; Humans; Hypochlorous Acid; Indicators and Reagents; Molsidomine; Nitrates; Nitrites; Nitrogen Oxides; Serum Albumin, Bovine; Sodium Nitrite; Spectrophotometry, Ultraviolet; Spermine; Tyrosine

1. Increased expression of inducible nitric oxide synthase (iNOS) and subsequent elevation of nitric oxide (NO) levels at inflammatory sites have led to the suggestion that peroxynitrite (the reaction product of superoxide and NO) is involved in pro-inflammatory processes. The present study has investigated the ability of peroxynitrite to induce oedema formation in the rat cutaneous microvasculature. 2. Peroxynitrite was synthesized from hydrogen peroxide and acidified nitrite. Spectrophotometry was used to measure the concentration and breakdown of peroxynitrite. It was also used to determine maximum amounts of hydrogen peroxide and sodium nitrite remaining after synthesis. 3. Oedema formation in response to intradermally (i.d.) injected peroxynitrite, hydrogen peroxide and sodium nitrite was measured by the extravascular accumulation of i.v. [125I]-albumin in the anaesthetized rat. 4. Peroxynitrite (40, 100 and 200 nmol/site) acted in a dose-dependent manner to cause a mean (+/- SEM) increase in plasma extravasation of 24 +/- 2, 55 +/- 5 and 69 +/- 6 microL, respectively (n = 4), with resulting inflammatory oedema. Peroxynitrite induced significantly larger plasma extravasation than equivalent vehicle controls at doses of 100 (P > 0.05) and 200 nmol (P > 0.001). This increased extravasation appears to be a direct microvascular response to peroxynitrite administration and not due to either a raised pH, necessary to stabilize the peroxynitrite, or contaminating concentrations of hydrogen peroxide or sodium nitrite from which peroxynitrite is formed. 5. These results suggest that peroxynitrite acts to increase microvascular permeability and oedema formation. Therefore, peroxynitrite may mediate vascular pro-inflammatory effects in addition to its direct cytotoxic activity.

Topics: Animals; Capillary Permeability; Hydrogen Peroxide; Hydrogen-Ion Concentration; Male; Nitrates; Rats; Rats, Wistar; Skin; Sodium Nitrite

Peroxynitrite anion is a powerful oxidant which can initiate nitration and hydroxylation of aromatic rings. Peroxynitrite can be formed in several ways, e.g. from the reaction of nitric oxide with superoxide or from hydrogen peroxide and nitrite at acidic pH. We investigated pH dependent nitration and hydroxylation resulting from the reaction of hydrogen peroxide and nitrite to determine if this reaction proceeds at pH values which are known to occur in vivo. Nitration and hydroxylation products of tyrosine and salicyclic acid were separated with an HPLC column and measured using ultraviolet and electrochemical detectors. These studies revealed that this reaction favored hydroxylation between pH 2 and pH 4, while nitration was predominant between pH 5 and pH 6. Peroxynitrite is presumed to be an intermediate in this reaction as the hydroxylation and nitration profiles of authentic peroxynitrite showed similar pH dependence. These findings indicate that hydrogen peroxide and nitrite interact at hydrogen ion concentrations present under some physiologic conditions. This interaction can initiate nitration and hydroxylation of aromatic molecules such as tyrosine residues and may thereby contribute to the biochemical and toxic effects of the molecules.

Topics: Hydrogen Peroxide; Hydrogen-Ion Concentration; Hydroxylation; Inflammation; Iron; Nitrates; Oxidative Stress; Salicylates; Salicylic Acid; Sodium Nitrite; Tyrosine

Topics: Artifacts; Drug Contamination; Hydrogen Peroxide; Hydrogen-Ion Concentration; Indicators and Reagents; Kinetics; Nitrates; Oxidation-Reduction; Oxygen; Sodium Nitrite

The vascular effect of peroxynitrite (ONOO-), a product of superoxide anion and nitric oxide, in isolated canine coronary arteries bathed in a 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered physiological salt solution (pH 7.4) was investigated. ONOO- was synthesized from nitrite and H2O2 in a quenched-flow reactor. Addition of 0.01 to 30 microM ONOO- produced a rapid, dose-dependent relaxation in all 17 rings of coronary arteries with a threshold concentration of 0.1 microM, IC50 of 1.0 +/- 0.1 microM and Emax of -96 +/- 0.5% (Means +/- S.E.M.). Incubation of arteries in a standard bicarbonate-buffered Krebs-Henseleit solution decreased slightly the sensitivity of ONOO- relaxation but did not alter the maximum effect (Emax = -97 +/- 1.1, n = 6 vessels). The ONOO(-)-induced coronary relaxation was reversible upon washing, and was also reproducible with repeated testings in the same ring. Mechanical removal of intimal endothelium did not alter the observed relaxant effect. Addition of superoxide dismutase (100 U/ml) potentiated the ONOO- relaxation by shifting the dose-response curve to the left (IC50 = 0.4 +/- 0.1 microM, P < .05, n = 17), whereas 3 microM hemoglobin inhibited it by shifting the curve to the right (IC50 = 20 +/- 4 microM, P < .05, n = 15). Relaxation was also observed with higher concentrations of sodium nitrite and decomposed ONOO-, although the time course of the development of these relaxations was considerably slower and with reduced sensitivity. In addition, superoxide dismutase had no effect on the latter relaxation responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arteries; Coronary Vessels; Dogs; Fluorescence; In Vitro Techniques; Light; Male; Nitrates; Nitric Oxide; Sodium Nitrite; Superoxides; Vasodilator Agents