Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle modified by peroxynitrite.

OBJECTIVE: Effect of peroxynitrite on SERCA1 activity was studied in correlation with enzyme carbonylation. Kinetic parameters and location of peroxynitrite effect on SERCA1 were determined. METHODS: Carbonyls were determined by immunoblotting. FITC, NCD-4 and Trp fluorescence were used to indicate changes in cytosolic and transmembrane regions of SERCA1. RESULTS: Peroxynitrite-concentration-dependent decrease of SERCA1 activity was associated with elevation of protein carbonyls. 4-HNE was not involved in carbonylation of SERCA1. Increased FITC fluorescence in the presence of peroxynitrite correlated with the decrease of the enzyme affinity to ATP. DISCUSSION AND CONCLUSION: Peroxynitrite-induced SERCA1 carbonylation that was not accompanied with the formation of 4-HNE-SERCA1 adducts is indicative of direct oxidation of SERCA1. As assessed by FITC fluorescence and decreased affinity of the enzyme to ATP, peroxynitrite impairment was found to occur in the cytosolic ATP-binding region of SERCA1.

Construction of a biomimetic peroxynitrite-generating platform: a two-component system to synthesize peroxynitrite in situ under the control of light.

Surge protector: a two-component peroxynitrite-generating platform has been engineered to release peroxynitrite (PN) in situ under the control of light. The system, which is constructed by layering sol-gel matrices containing xanthine oxidase (bottom layer) and a metal nitrosyl (top layer), allows studies of PN chemistry at varying fluxes of its precursors.

Visualization of peroxynitrite-induced changes of labile Zn2+ in the endoplasmic reticulum with benzoresorufin-based fluorescent probes.

Zn(2+) plays essential roles in biology, and the homeostasis of Zn(2+) is tightly regulated in all cells. Subcellular distribution and trafficking of labile Zn(2+), and its inter-relation with reactive nitrogen species, are poorly understood due to the scarcity of appropriate imaging tools. We report a new family of red-emitting fluorescent sensors for labile Zn(2+), ZBR1-3, based on a benzoresorufin platform functionalized with dipicolylamine or picolylamine-derived metal binding groups. In combination, the pendant amines and fluorophore afford an [N3O] binding motif that resembles that of previously reported fluorescein-based sensors of the Zinpyr family, reproducing well their binding capabilities and yielding comparable Kd values in the sub-nanomolar and picomolar ranges. The ZBR sensors display up to 8.4-fold emission fluorescence enhancement upon Zn(2+) binding in the cuvette, with similar responses obtained in live cells using standard wide-field fluorescence microscopy imaging. The new sensors localize spontaneously in the endoplasmic reticulum (ER) of various tested cell lines, allowing for organelle-specific monitoring of zinc levels in live cells. Study of ER zinc levels in neural stem cells treated with a peroxynitrite generator, Sin-1, revealed an immediate decrease in labile Zn(2+) thus providing evidence for a direct connection between ER stress and ER Zn(2+) homeostasis.

Refined representation of falloff curves for the reaction HO + NO2 + N2 → (HONO2, HOONO) + N2.

Experimental data for the reactions (1) HO + NO(2) (+N(2)) → HONO(2) (+N(2)) and (2) HO + NO(2) (+N(2)) → HOONO (+N(2)) near 300 K and over the pressure range 1 Torr to 320 bar are represented in terms of novel asymmetric broadening factors in falloff expressions. This analysis allows for a refined representation of the data, reproducing fine details of k = k(1) + k(2) and k(2)/k(1) and probably allows for a better extrapolation to the limiting low and high pressure rate constants than possible with symmetric broadening factors in conventional falloff expressions. The experimental data clearly show that the center broadening factor F(cent,1) is close to 0.41 and consistent with results from theoretical modeling. This value of F(cent) markedly differs from the "standard value" of 0.6, and the consequences of this difference are illustrated. Limiting rate constants of k(1,0) = [N(2)] (T/300 K)(-4.5) 3.2 × 10(-30) cm(6) molecule(-2) s(-1), k(2,0) = [N(2)] (T/300 K)(-4.5) 1.0 × 10(-31) cm(6) molecule(-2) s(-1), k(1,∞) = 2.7 × 10(-11) cm(3) molecule(-1) s(-1), and k(2,∞) = 4.8 × 10(-11) cm(3) molecule(-1) s(-1) are obtained and tested over the range 220-300 K, whereas the exponent -4.5 changes to -3.0 in k(1,0) and k(2,0) over the range 300-430 K (the values correspond to falloff curves with asymmetric broadening factors).

GC-MS and HPLC methods for peroxynitrite (ONOO- and O15NOO-) analysis: a study on stability, decomposition to nitrite and nitrate, laboratory synthesis, and formation of peroxynitrite from S-nitrosoglutathione (GSNO) and KO2.

Nitric oxide (˙NO) and superoxide (O(2)(-)˙) are ubiquitous in nature. Their reaction product peroxynitrite (ONOO(-)) and notably its conjugated peroxynitrous acid (ONOOH) are highly unstable in aqueous phase. ONOO(-)/ONOOH (referred to as peroxynitrite) isomerize and decompose to NO(3)(-), NO(2)(-) and O(2). Here, we report for the first time GC-MS and HPLC methods for the analysis of peroxynitrite in aqueous solution. For GC-MS analysis peroxynitrite in alkaline solution was derivatized to a pentafluorobenzyl derivative using pentafluorobenzyl bromide. O(15)NOO(-) was synthesized from H(2)O(2) and (15)NO(2)(-) and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil® 100-5C(18)AB or Nucleodur® C(18) Gravity. The mobile phase consisted of a 10 mM aqueous solution of tetrabutylammonium hydrogen sulfate and had a pH value of 11.5. UV absorbance detection at 300 nm was used. HPLC allows simultaneous analysis of ONOO(-), NO(2)(-) and NO(3)(-). The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[(15)N]nitrosoglutathione (GS(15)NO) and KO(2), and isomerization/decomposition of peroxynitrite to NO(2)(-) and NO(3)(-) in aqueous buffer.

In vitro effects of peroxynitrite treatment on fish liver catalase activity.

The effect of peroxynitrite (PN), a highly toxic agent, on catalase (CAT) activity in fish liver microsomal homogenates was determined. PN was synthesized by mixing acidic hydrogen peroxide solution with sodium nitrite solution and then adding sodium hydroxide solution into the mixture in order to stabilize the highly labile compound peroxynitrous acid (ONOOH) in peroxynitrite anion form (ONOO(- )). The effect of PN and decomposed peroxynitrite (DPN), prepared by preincubation with HCl, was monitored by using a constant amount of homogenate containing the CAT enzyme. Significant losses were observed in the CAT activity of fish liver enzyme after treatment with PN and also with DPN products, the inhibitory effect of PN being slightly more pronounced than that of DPN. IC(50) values were 5.5 and 8.5 microM for PN and DPN, respectively. The PN inhibition of CAT activity is due to both the effects of the secondary and decomposition products of PN and its nitration and oxidation effects on the amino acid residues of the enzyme.

A new hammer in the redox toolbox: high-purity peroxynitrite for cell signaling and toxicology studies.

A new chemical synthesis has been disclosed for the oxidizing and nitrating species peroxynitrite. This nitric oxide and superoxide-derived cell signaling mediator can also serve as a toxicant at higher rates of generation. The new synthetic strategy reported by Sturzbecher-Höhne and colleagues overcomes long-standing issues with purity and quality of peroxynitrite preparations and should lay a foundation for nailing new insight into the redox reactions of peroxynitrite and its products.

The bactericidal and morphological effects of peroxynitrite on Helicobacter pylori.

Peroxynitrite (ONOO-) is correlated with the pathogenesis of Helicobacter pylori-induced peptic ulcer diseases. We aimed to investigate the time- and concentration-dependent bactericidal and morphological effects of ONOO- on H. pylori. Authentic ONOO- was synthesized as quenched-flow method. A stock culture of H. pylori NCTC 11637 was exposed to different concentrations of ONOO- (0.1-40 micromol/L) or decomposed ONOO- or fresh medium. Samples were taken at 0, 15, 30, 60, and 120 minutes, for the evaluation of viable bacteria and bacterial morphology with gram strain and transmission electron microscopy. Decomposed ONOO- showed no bactericidal activity against H. pylori. ONOO- application caused a decrease in the number of viable bacteria within the first 15 minutes. The significant conversion of H. pylori from spiral form to coccoid form was determined with 10 micromol/L of ONOO-, and higher concentrations caused lysis of the cells. Separation of cell wall, bleb formation, vacuolization, decrease of secretory granules, and lysis of bacteria were the morphological effects of ONOO- on H. pylori. Because the morphology of the bacteria is one of the important factors in virulence; peroxynitrite-related morphological effects might have an impact in the progress of the H. pylori-induced peptic ulcer diseases.

Preparation and properties of lithium and sodium peroxynitrite.

We describe the preparation of aqueous solutions of LiONOO and NaONOO from (Me4N)ONOO. An aqueous solution of analytically pure, commercially available (Me4N)ONOO is applied to an Amberlyst 15 column at 4 degrees C, and the Me4N+ is rapidly (in 20 min) exchanged against Li+ or Na+. The exchanged peroxynitrite is produced in yields of ca. 90% with a nitrite content of 5-6%.

Inhibition of PTEN by peroxynitrite activates the phosphoinositide-3-kinase/Akt neuroprotective signaling pathway.

Peroxynitrite is usually considered as a neurotoxic nitric oxide-derivative. However, an increasing body of evidence suggests that, at low concentrations, peroxynitrite affords transient cytoprotection, both in vitro and in vivo. Here, we addressed the signaling mechanism responsible for this effect, and found that rat cortical neurons in primary culture acutely exposed to peroxynitrite (0.1 mmol/L) rapidly elicited Akt-Ser(473) phosphorylation. Inhibition of phosphoinositide-3-kinase (PI3K)/Akt pathway with wortmannin or Akt small hairpin RNA (shRNA) abolished the ability of peroxynitrite to prevent etoposide-induced apoptotic death. Endogenous peroxynitrite formation by short-term incubation of neurons with glutamate stimulated Akt-Ser(473) phosphorylation, whereas Akt shRNA enhanced the vulnerability of neurons against glutamate. We further show that Akt-Ser(473) phosphorylation was consequence of the oxidizing, but not the nitrating properties of peroxynitrite. Peroxynitrite failed to nitrate or phosphorylate neurotrophin tyrosine kinase receptors (Trks), and it did not modify the ability of brain-derived neurotrophic factor (BDNF), to phosphorylate its cognate receptor, TrkB; however, peroxynitrite enhanced BDNF-mediated Akt-Ser(473) phosphorylation. Finally, we found that peroxynitrite-stimulated Akt-Ser(473) phosphorylation was associated with an increased proportion of oxidized phosphoinositide phosphatase, PTEN, in neurons. Moreover, peroxynitrite prevented the increase of apoptotic neuronal death caused by over-expression of PTEN. Thus, peroxynitrite exerts neuroprotection by inhibiting PTEN, hence activating the anti-apoptotic PI3K/Akt pathway in primary neurons.

The degradative action of peroxynitrite on high-molecular-weight hyaluronan.

OBJECTIVES: This contribution presents the results of the kinetics of HA degradation by peroxynitrite, which represents one of the main reactive oxygen species degrading various biomacromolecules under inflammatory conditions. METHODS: Two simple procedures have been adapted to prepare sodium peroxynitrite: the first containing an excess of H(2)O(2), and the second in which the H(2)O(2) excess had been decomposed by MnO(2) treatment. The kinetics of hyaluronan degradation by action of peroxynitrite was monitored by rotational viscometry. RESULTS: High-molecular-weight hyaluronan was degraded by peroxynitrite. The degradation was increased in the presence of ONOO(-) previously treated by MnO(2) in order to remove residual hydrogen peroxide. One of the reasons of this finding could be that by the action of the residual metal the pathway of ONOO(-) decomposition starts to be manifested immediately on mixing traces of metals originally present in the HA sample with the ions of manganese. CONCLUSIONS: Trace amounts of transition metal(s) should be taken into consideration on evaluating the experimental results. Purchase of the marketed peroxynitrite product appears to be the appropriate approach to simplify and standardize the quality of ONOONa.

Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism.

Antioxidant activities of 15 purified bilberry anthocyanins together with pelargonidin 3-O-beta-D-glucopyranoside and 4'-O-methyl delphinidin 3-O-beta-D-glucopyranoside (MDp 3-glc), the major metabolite of delphinidin 3-O-beta-D-glucopyranoside (Dp 3-glc), were evaluated in order to study the structure-antioxidant activity relationship and any synergism among them in the mixture. Both aglycone structure and the attached sugar moiety affected the O*2- and ONOO- -scavenging activities, although the effect of the attached sugar moiety was smaller than that of the aglycone structure. The potency of activity toward the superoxide radical was in the following order: delphinidin > petunidin > malvidin =approximately cyanidin>(+)-catechin > peonidin > pelargonidin. The activity toward ONOO- was: delphinidin > cyanidin =approximately petunidin > malvidin =approximately (+)-catechin > peonidin > pelargonidin. It was confirmed that methylation of 4'-OH markedly reduced the antioxidant activity of anthocyanin. Further, it was revealed that synergism occurred in both - and ONOO- -scavenging activities among the anthocyanins in the mixture.

Oxidative and nitrative modifications of enkephalins by reactive nitrogen species.

The interaction of Leucine-enkephalin (Leu-enkephalin) with reactive nitrogen species has been investigated. Reactive nitrogen species are capable of nitrating and oxidizing Leu-enkephalin. HPLC analysis shows the formation of two major enkephalin derivatives by peroxynitrite. The tyrosine amino-terminal residue of Leu-enkephalin is converted either to 3-nitrotyrosine thus producing nitroenkephalin and to dityrosine by dimerization with the production of an enkephalin dimer. The evidence of the formation of the nitroenkephalin and of the enkephalin dimer--dienkephalin--was achieved by electrospray ionisation mass spectrometry. In addition to peroxynitrite, the methylene blue photosensitized oxidation of enkephalin in the presence of nitrite leads to the formation of the nitrated peptide. Moreover, the nitropeptide can be also obtained by peroxidase-generated nitrogen reactive species.

Synthesis of peroxynitrite using isoamyl nitrite and hydrogen peroxide in a homogeneous solvent system.

A method for the synthesis of peroxynitrite is described. It involves nitrosation of H2O2 at pH> or = 12.5 by isoamyl or butyl nitrite in mixed solvents of isopropyl alcohol (IPA) and water at 25+/-1 degrees C. Maximum yields of peroxynitrite are obtained after 15 min of incubation at IPA concentrations of 30-70% (v/v). The solutions of peroxynitrite are processed for removal of IPA and isoamyl alcohol by solvent extraction. Unreacted H2O2 is removed by catalytic decomposition on granular MnO(2). The post processed solutions of peroxynitrite are useful in several chemical and biochemical investigations where bolus additions are required. The method as reported is amenable for large scale synthesis as it involves sequential mixing of solvents (water and IPA) to alkali followed by the addition of H2O2 and alkyl nitrite.

Synthesis of peroxynitrite from nitrite and hydrogen peroxide.

We report a simple method for the synthesis of peroxynitrite from nitrite and hydrogen peroxide that can generate hundreds of milliliters of 180 mM peroxynitrite within 1 h from start to finish. It can be scaled down to make small quantities of isotope-labeled peroxynitrite. The method requires only a syringe pump and tubing connectors and is feasible for any biochemical laboratory. Unreacted hydrogen peroxide is eliminated with manganese dioxide, using an improved preparation compared to commercially available manganese dioxide. A number of contaminants were detected by mass spectrometry in peroxynitrite solutions cleaned with commercially purchased manganese dioxide. Nitrite contamination of the peroxynitrite solution is less than 2% as determined using the Griess method. The residual contaminants are principally 0.28 M sodium chloride and 0.1 M sodium hydroxide, which pose few problems when peroxynitrite is diluted for use in biological experiments.

Generation of peroxynitrite from reaction of N-acetyl-N-nitrosotryptophan with hydrogen peroxide over a wide range of pH values.

The novel reaction of N-acetyl-N-nitrosotryptophan (NANT) with hydrogen peroxide to yield peroxynitrite is demonstrated. Quantum chemical calculations performed at CBS-QB3 level of theory predicted that the reaction of N-nitrosoindole with both H(2)O(2) and its corresponding anion is thermodynamically feasible. At pH 13, the formation of peroxynitrite from the bimolecular reaction of NANT with H(2)O(2) is unequivocally demonstrated by (15)N NMR spectrometry. In order to prove the intermediacy of peroxynitrite from the NANT-H(2)O(2) system at neutral (7.4) and acidic pH (4.5), the characteristic pattern of CIDNP (chemically induced dynamic nuclear polarization) signals were recorded, i.e. enhanced absorption in the (15)N NMR signal of nitrate and emission in the (15)N NMR signal of nitrite. Most interestingly, the NANT-H(2)O(2) system nitrated N-acetyltyrosine at pH 4 via recombination of freely diffusing nitrogen dioxide and tyrosyl radicals, but nitration was negligible at pH 7.4. Since the combination between NANT and H(2)O(2) is slow, endogenous N-nitrosotryptophan residues cannot act as a "carrier" for peroxynitrite.

Fluorescence measurements of steady state peroxynitrite production upon SIN-1 decomposition: NADH versus dihydrodichlorofluorescein and dihydrorhodamine 123.

The production of peroxynitrite during 3-morpholinosydnonimine (SIN-1) decomposition can be continuously monitored, with a sensitivity < or = 0.1 microM, from the kinetics of NADH fluorescence quenching in phosphate buffers, as well as in buffers commonly used with cell cultures, like Locke's buffer or Dulbecco's modified Eagle's medium (DMEM-F12). The half-time for peroxynitrite production during SIN-1 decomposition ranged from 14-18 min in DMEM-F12 (plus and minus phenol red) to 21.5 min in Locke's buffer and 26 min in DMEM-F12 supplemented with apotransferrin (0.1 mg/mL). The concentration of peroxynitrite reached a peak that was linearly dependent upon SIN-1 concentration, and that for 100 microM SIN-1 amounted to 1.4 +/- 0.2 microM in Locke's buffer, 3.2-3.6 microM in DMEM-F12 (plus and minus phenol red) and 1.8 microM in DMEM-F12 supplemented with apotransferrin. Thus, the maximum concentration of peroxynitrite ranged from 1.2 to 3.6% of added SIN-1. NADH was found to be less sensitive than dihydrorhodamine 123 and 2',7'-dichlorodihydrofluorescein diacetate to oxidation by H2O2, which is produced during SIN-1 decomposition in common buffers. It is shown that peroxynitrite concentration can be controlled (+/-5%) during predetermined times by using sequential SIN-1 pulses, to simulate chronic exposure of cells or subcellular components to peroxynitrite.

Protection by flavonoids against the peroxynitrite-mediated oxidation of dihydrorhodamine.

Peroxynitrite anion is a reactive and short-lived species and its formation in vivo has been implicated in several human diseases. In view of the potential usefulness of compounds that can protect against peroxynitrite or their reactive intermediates, a study focused on flavonoid compounds was carried out. Since the reactivity of peroxynitrite may be modified by Co2/HCO3-, which is an important plasma buffer, the protection of flavonoids against peroxynitrite was evaluated by their ability to inhibit the peroxynitrite-mediated dihydrorhodamine (DHR123) oxidation with or without physiological concentrations of bicarbonate. Flavonoids from different classes were studied to elucidate which structural features are required for an effective protection. The most efficient flavonoids on protecting DHR123 against oxidation by peroxynitrite have their ability diminished in the presence of bicarbonate, but they maintain the hierarchy established in the absence of bicarbonate. The flavones are the most effective flavonoids and their effects depend mainly on the number of hydroxyl groups. These must include either a catechol group in the B-ring or a hydroxyl group at the 3-position. This work also included some isoflavones, flavanones and a flavanol, which enable us to conclude about the importance of another structural feature: the 2,3-double bond. These results indicate that the ability of flavonoids to protect against peroxynitrite depends on some structural features, also important to scavenge oxygen free radicals and to chelate metal ions. The most efficient flavonoids are effective at low concentrations with IC50 of the same magnitude as Ebselen, a selenocompound that has been reported to be excellent at protecting against peroxynitrite. Their effectiveness at low concentrations is an important aspect to take into account when characterizing a compound as an antioxidant with biological interest.

Determination of pipemidic acid based on flow-injection chemiluminescence due to energy transfer from peroxynitrous acid synthesized on-line.

A flow-injection chemiluminescence (CL) method for the determination of pipemidic acid is described. It is based on energy transfer from excited state peroxynitrous acid to pipemidic acid, in which the excited state peroxynitrous acid is synthesized on-line by the mixing of acid hydrogen peroxide with nitrite in a flow system and the CL is from two excited states of pipemidic acid. The proposed method allows the measurement of pipemidic acid over the range of 2.0 x 10(-7)-2.0 x 10(-5) mol l(-1) . The detection limit is 6.3 x 10(-8) mol l(-1), and the relative standard deviation for 2.0 x 10(-6) mol l(-1) pipemidic acid (n = 9) is 0.9%. This method was evaluated by the analysis of pipemidic acid in pharmaceutical preparations.

Peroxynitrite delivery methods for toxicity studies.

The endogenous synthesis of peroxynitrite (ONOO(-)) has been implicated in a number of diseases, but assessments of its cytotoxicity and genotoxicity have been hampered by its extremely short half-life under physiological conditions (<20 ms) and the consequent difficulty in exposing cells to known concentrations of it over at least several hours. Two methods for peroxynitrite delivery to cell cultures were investigated, one involving steady infusion of preformed ONOO(-) and the other based on the continuous in situ synthesis of ONOO(-) from NO and O(2)(-). In the latter, NO was supplied by diffusion through gas permeable tubing and O(2)(-) was generated using the hypoxanthine-xanthine oxidase reaction. The performance of both methods was assessed by measuring the rates of formation of tyrosine derivatives (dityrosine and nitrotyrosine) that are commonly employed as biomarkers for peroxynitrite. Experimental results in the absence of cells were compared in each case with predictions from kinetic models. In the infusion system, the measured dityrosine and nitrotyrosine yields were in excellent agreement with those predicted from the model. To characterize the other system, experiments were performed first to determine the kinetics of hypoxanthine oxidation by xanthine oxidase and uric acid oxidation by uricase. Simulations of the complex reaction network in the complete synthesis system suggested that dityrosine should be the major product there, that the yields of both tyrosine derivatives should be very sensitive to the relative rates of NO and O(2)(-) delivery, and that equal rates for NO and O(2)(-) should maximize those yields. Experiments performed under the predicted optimal conditions showed much lower levels of dityrosine than expected and no detectable nitrotyrosine. The unexpectedly low yields of tyrosine products could be explained largely by the partial inactivation of both xanthine oxidase and uricase by peroxynitrite-derived NO(2) and CO(3)(-) radicals. We conclude that continuous infusion of peroxynitrite is the more promising approach.