Authentic ONOO Research Articles

Novel Boronate Probe Based on 3-Benzothiazol-2-yl-7-hydroxy-chromen-2-one for the Detection of Peroxynitrite and Hypochlorite.

Derivatives of coumarin, containing oxidant-sensitive boronate group, were recently developed for fluorescent detection of inflammatory oxidants. Here, we report the synthesis and the characterization of 3-(2-benzothiazolyl)-7-coumarin boronic acid pinacol ester (BC-BE) as a fluorescent probe for the detection of peroxynitrite (ONOO–), with high stability and a fast response time. The BC-BE probe hydrolyzes in phosphate buffer to 3-(2-benzothiazolyl)-7-coumarin boronic acid (BC-BA) which is stable in the solution even after a prolonged incubation time (24 h). BC-BA is slowly oxidized by H2O2 to form the phenolic product, 3-benzothiazol-2-yl-7-hydroxy-chromen-2-one (BC-OH). On the other hand, the BC-BA probe reacts rapidly with ONOO−. The ability of the BC-BA probe to detect ONOO– was measured using both authentic ONOO– and the system co-generating steady-state fluxes of O2•– and •NO. BC-BA is oxidized by ONOO– to BC-OH. However, in this reaction 3-benzothiazol-2-yl-chromen-2-one (BC-H) is formed in the minor pathway, as a peroxynitrite-specific product. BC-OH is also formed in the reaction of BC-BA with HOCl, and subsequent reaction of BC-OH with HOCl leads to the formation of a chlorinated phenolic product, which could be used as a specific product for HOCl. We conclude that BC-BA shows potential as an improved fluorescent probe for the detection of peroxynitrite and hypochlorite in biological settings. Complementation of the fluorescence measurements by HPLC-based identification of oxidant-specific products will help to identify the oxidants detected.

Redox Regulation of Methylation by PRMT1

ADMA and L‐NMMA are endogenous NOS inhibitors derived from the proteolysis of methylated arginine residues on proteins. The methylation is carried out by a group of enzymes referred to as protein‐arginine methyl transferase's (PRMTs). Protein arginine methylation has been demonstrated to regulate key cellular events including transcription, RNA metabolism and protein‐protein interactions and recent evidence suggest that PRMT activity may be under redox control. Therefore, the aim of the current study was to assess the redox‐dependent regulation of PRMT1 activity using recombinant proteins. Results demonstrated a dose dependent increase in PRMT activity in the presence of NO donor DETANONOate (0.1 nM‐1 mM) with a maximal increase of 300% at 1 mM. In contrast, exposure of purified PRMT1 to authentic H2O2 (0.1 nM‐1 mM) and ONOO− (0.1 nM‐1 mM) resulted in decreased PRMT activity with 90% inhibition observed at 1 mM ONOO−. Peroxynitrite inhibition of PRMT1 was accompanied by the formation of nitro‐tyrosine residues on PRMT1 suggesting that tyrosine nitration events are responsible for the inhibition. Increased activity of PRMT1 by exposure to NO would be expected to result in increased formation of the competitive eNOS inhibitors ADMA and L‐NMMA. Thus, NO may form a negative feedback mechanism to regulate activity of eNOS by increasing the activity of PRMT1 and therefore escalating the production of ADMA and L‐NMMA.

Selective peroxynitrite scavenging activity of 3-methyl-1,2-cyclopentanedione from coffee extract.

It has been known that reactive oxygen and nitrogen species such as nitric oxide (NO), superoxide radical (*O2-) and their byproduct peroxynitrite (ONOO-) induce cellular and tissue injury, ultimately resulting in several human diseases. In this study, we examined scavenging effects of 3-methyl-1,2-cyclopentanedione (MCP) from coffee extract on the reactivity of those toxic molecules. MCP significantly inhibited both the oxidation of 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) by reactive oxygen species (ROS) (mainly *O2-) from kidney homogenate (41% at 100 microM) and the generation of fluorescent 4,5-diaminofluorescein (DAF-2) by NO from sodium nitroprusside (IC50 (concn producing 50% inhibition), 63.8 microM). More potently, however, MCP suppressed the oxidation of dihydrorhodamine 123 (DHR 123) to fluorescent rhodamine 123 mediated by authentic ONOO- with an IC50 value of 3.3 microM. The neutralizing effect of the reactivity of ONOO- by MCP was due to electron donation, not nitration of the compound. Additionally, MCP also decreased ONOO- formation of nitrotyrosine adducts of glutathione (GSH) reductase, and consequently protected the enzyme activity of GSH reductase against decreasing by ONOO-, indicating that MCP may prevent ONOO- -induced damage of GSH reductase. Furthermore, MCP only weakly suppressed NO production, which is one of the upstream sources of ONOO- in-vivo, suggesting that NO production may be not a pharmacological target for MCP. Taken together, our results suggest that MCP may be regarded as a selective regulator of ONOO- -mediated diseases via direct scavenging activity of ONOO-.

Evaluation of Salicornia herbacea as a Potential Antioxidant and Anti-Inflammatory Agent

In this study, the antioxidant and anti-inflammatory activities of Salicornia herbacea were evaluated. The crude CH(2)Cl(2)/methanol extract of S. herbacea showed 52% and 86% scavenging activities of the authentic ONOO(-) and ONOO(-) from 3-morpholinosydnomimine (SIN-1) at a concentration of 50 microg/mL, respectively, and was subjected to a further fractionation with n-hexane, 85% aqueous methanol, n-butanol, and water. Additional purification of the n-butanol fraction revealed that the most potent scavenging activity led to the isolation of isorhamnetin 3-O-beta-d-glucopyranoside as the active principle. The structure of isorhamnetin 3-O-beta-d-glucopyranoside was elucidated by extensive two-dimensional nuclear magnetic resonance experiments such as (1)H correlation spectroscopy nuclear Overhauser effect spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple-bond correlation as well as by comparison with the published spectral data. Isorhamnetin 3-O-beta-d-glucopyranoside exhibited dose-dependent scavenging activities of the authentic ONOO(-) and ONOO(-) from SIN-1. The electron spin resonance spin-trap techniques confirmed that reactive oxygen species, including the hydroxyl, superoxide, carbon-centered, and 1,1-diphenyl-2-picrylhydrazyl radicals, were actively quenched by addition of isorhamnetin 3-O-beta-d-glucopyranoside. In addition, isorhamnetin 3-O-beta-d-glucopyranoside suppressed the lipopolysaccharide-induced nitric oxide production and the expression of cytokines such as inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-1beta in Raw 264.7 cells. Findings from this study should underscore the nutraceutical value of S. herbacea-derived isorhamnetin 3-O-beta-d-glucopyranoside as a potent antioxidative and anti-inflammatory agent via alleviation of radical-induced toxicities and pro-inflammatory responses.

Regulation of the soluble epoxide hydrolase by peroxynitrite

Inhibition of the soluble epoxide hydrolase (sEH), which for example converts epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids (DHETs), has anti‐inflammatory as well as anti‐hypertensive effects. As the enzymatic core of the sEH hydrolase domain contains two tyrosine residues (Tyr383 & 466), we addressed the hypothesis that the activity of the sEH may be affected by oxidative stress, in particular by nitration of Tyr383 & 466.sEH activity was measured in human and murine endothelial cells by LC‐MS/MS and was sensitive to the specific sEH inhibitor 1‐adamantyl‐3‐cyclohexylurea. The importance of the tyrosine residues was demonstrated in that mutation of Tyr383 & 466 to phenylalanine resulted in a complete loss of DHET production. sEH activity in both cell types was significantly (80±6%, P<0.001) attenuated by preincubation with the peroxynitrite (ONOO−) generator 3‐morpholino‐sydnonimine (SIN‐1, 0.5 mmol/L). The latter compound and authentic ONOO− also elicited the tyrosine nitration of the sEH (immunoprecipitation and Western blotting) an effect that was not be triggered by hydrogen peroxide (1 mmol/L). Moreover, in an in vivo murine model, oxidative stress induced by bacterial lipopolysaccharide significantly (20±9%, P<0.01) attenuated lung sEH activity. These data indicate that the activity of the sEH and thus cellular EET levels can be regulated by the tyrosine nitration of the protein.

Nitric Oxide Evokes an Adaptive Response to Oxidative Stress by Arresting Respiration

Aerobic metabolism generates biologically challenging reactive oxygen species (ROS) by the endogenous autooxidation of components of the electron transport chain (ETC). Basal levels of oxidative stress can dramatically rise upon activation of the NADPH oxidase-dependent respiratory burst. To minimize ROS toxicity, prokaryotic and eukaryotic organisms express a battery of low-molecular-weight thiol scavengers, a legion of detoxifying catalases, peroxidases, and superoxide dismutases, as well as a variety of repair systems. We present herein blockage of bacterial respiration as a novel strategy that helps the intracellular pathogen Salmonella survive extreme oxidative stress conditions. A Salmonella strain bearing mutations in complex I NADH dehydrogenases is refractory to the early NADPH oxidase-dependent antimicrobial activity of IFNgamma-activated macrophages. The ability of NADH-rich, complex I-deficient Salmonella to survive oxidative stress is associated with resistance to peroxynitrite (ONOO(-)) and hydrogen peroxide (H(2)O(2)). Inhibition of respiration with nitric oxide (NO) also triggered a protective adaptive response against oxidative stress. Expression of the NDH-II dehydrogenase decreases NADH levels, thereby abrogating resistance of NO-adapted Salmonella to H(2)O(2). NADH antagonizes the hydroxyl radical (OH(.)) generated in classical Fenton chemistry or spontaneous decomposition of peroxynitrous acid (ONOOH), while fueling AhpCF alkylhydroperoxidase. Together, these findings identify the accumulation of NADH following the NO-mediated inhibition of Salmonella's ETC as a novel antioxidant strategy. NO-dependent respiratory arrest may help mitochondria and a plethora of organisms cope with oxidative stress engendered in situations as diverse as aerobic respiration, ischemia reperfusion, and inflammation.

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.

Degradation of matrix glycosaminoglycans by peroxynitrite/peroxynitrous acid: Evidence for a hydroxyl-radical-like mechanism

The oxidant peroxynitrite/peroxynitrous acid (ONOO −/ONOOH) is generated at sites of inflammation via reaction of O 2 − with NO. Previous studies have shown that these species can oxidize cellular targets, but few data are available on damage to extracellular matrix and its components, despite evidence for matrix modification in a number of pathologies. In the current study we show that reaction of ONOO −/ONOOH with glycosaminoglycans results in extensive polymer fragmentation. Bolus authentic ONOO −/ONOOH modifies hyaluronan, heparin, and chondroitin, dermatan, and heparan sulfates, in a concentration-dependent, but O 2-independent, manner. The ONOO −/ONOOH generator 3-(4-morpholinyl)sydnoneimine produces similar time- and concentration-dependent damage. These reactions generate specific polymer fragments via cleavage at disaccharide intervals. Studies at different pH values, and in the presence of bicarbonate, are consistent with ONOOH, rather than the carbonate adduct, CO 3 − or ONOO −, being the source of damage. EPR spin trapping experiments have provided evidence for the formation of carbon-centered radicals on glycosaminoglycans and related monosaccharides; the similarity of these spectra to those obtained with authentic HO is consistent with fragmentation being induced by this oxidant. These data suggest that extracellular matrix fragmentation at sites of inflammation may be due, in part, to the formation and reactions of ONOOH.

In vitro peroxynitrite scavenging activity of 6-hydroxykynurenic acid and other flavonoids fromGingko biloba yellow leaves

As part of our research on phytochemicals that exert protective effects against diseases related to reactive nitrogen species, we have evaluated the scavenging activity of the yellow leaves of Ginkgo biloba on ONOO-. The methanol extract and ethyl acetate fraction obtained from yellow leaves of G. biloba evidenced a marked scavenging activity on authentic ONOO-. Repeated column chromatography of the active ethyl acetate soluble fraction on silica gel, Sephadex LH-20, and RP-18, resulted in the purification of 15 known compounds, including sciadopitysin (1), ginkgolide B (2), bilobalide (3), isoginkgetin (4), kaempferol (5), luteolin (6), protocatechuic acid (7), bilobetin (8), amentoflavone (9), beta-sitosterol glucopyranoside (10), kaempferol 3-O-rhamnopyranoside (11), kaempferol 3-O-glucopyranoside (12), kaempferol 3-O-[6"'-O-p-coumaroyl-beta-D-glucopyranosyl(1 --> 2)-alpha-L-rhamnopyranoside] (13), kaempferol 3-O-rutinoside (14), and 6-hydroxykynurenic acid (15). Among the compounds isolated, flavonoids (5, 6 and 11-14), protocatechuic acid (7), and 6-hydroxykynurenic acid (15) all exhibited marked scavenging activities on authentic ONOO-. The IC50 values of 5-7, 11-14 and 15 were as follows: 2.86 +/- 0.70, 2.30 +/- 0.04, 2.85 +/- 0.10, 5.60 +/- 0.47, 4.16 +/- 1.65, 2.47 +/- 0.15, 3.02 +/- 0.48, and 6.24 +/- 0.27 microM, respectively. DL-Penicillamine (IC50 = 4.98 +/- 0.27 microM) was utilized as a positive control. However, the other compounds (1-4, 8-10) exerted no effects against ONOO-.

In Vitro Free Radical and ONOO- Scavengers from Sophora flavescens

Activity-guided fractionation of the CH2Cl2-soluble fraction of the roots of Sophora flavescens furnished five 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavengers: trans-hexadecyl ferulic acid (1), cis-octadecyl ferulic acid (2), trans-hexadecyl sinapic acid (3), (-)-4-hydroxy-3-methoxy-(6aR,11aR)-8,9-methylenedioxypterocarpan (4) and desmethylanhydroicaritin (8), along with nine known inactive compounds: (-)-maackiain (5), xanthohumol (6), formononetin (7), (2S)-2'-methoxykurarinone (9), (2S)-3beta,7,4'-trihydroxy-5-methoxy-8-(gamma,gamma-dimethylallyl)-flavanone (10), (2S)-7,4'-dihydroxy-5-methoxy-8-(gamma,gamma-dimethylallyl)-flavanone (11), umbelliferone (12), kuraridin (13), and trifolirhizin (14). Compounds 1-4 and 8 exhibited DPPH free radical scavenging effects at IC50 values of 33.01 +/- 0.20, 57.06 +/- 0.16, 39.84 +/- 0.36, 35.83 +/- 0.47, and 18.11 +/- 0.04 microM, respectively. L-Ascorbic acid, when used as a positive control, exhibited an IC50 value of 7.39 +/- 0.01 microM. Compounds 1-4 and 8 also appeared to exert significant scavenging effects on authentic ONOO-, with IC50 values of 5.76 +/- 1.19, 15.06 +/- 1.64, 8.17 +/- 4.97, 1.95 +/- 0.29, and 4.06 +/- 2.41 microM, respectively. Penicillamine (IC50 = 2.36 +/- 0.79 microM) was used as a positive control. In addition, compounds 2, 4, 6, 8, and 10 were isolated from this plant for the first time.

Protective effects of Asian green vegetables against oxidant induced cytotoxicity

To evaluate the antioxidant and phase II detoxification enzyme inducing ability of green leaf vegetables consumed in Asia. The antioxidant properties of six commonly consumed Asian vegetables were determined using the ABTS, DPPH, deoxyribose, PR bleaching and iron- ascorbate induced lipid peroxidation assay. Induce of phase II detoxification enzymes was also determined for each respective vegetable extract. Protection against authentic ONOO- and HOCl mediated cytotoxicity in human colon HCT116 cells was determined using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) viability assay. All of the extracts derived from green leaf vegetables exhibited antioxidant properties, while also having cytoprotective effects against ONOO- and HOCl mediated cytotoxicity. In addition, evaluation of the phase II enzyme inducing ability of each extract, as assessed by quinone reductase and glutathione-S-transferase activities, showed significant variation between the vegetables analyzed. Green leaf vegetables are potential sources of antioxidants and phase II detoxification enzyme inducers in the Asian diet. It is likely that consumption of such vegetables is a major source of beneficial phytochemical constituents that may protect against colonic damage.

Hesperetin: A Potent Antioxidant Against Peroxynitrite

Peroxynitrite (ONOO-) is a reactive oxidant formed from superoxide (•O2-) and nitric oxide (•NO), that can oxidize several cellular components, including essential protein, non-protein thiols, DNA, low-density lipoproteins (LDL), and membrane phospholipids. ONOO- has contributed to the pathogenesis of diseases such as stroke, heart disease, Alzheimer's disease, and atherosclerosis. Because of the lack of endogenous enzymes to thwart ONOO- activation, developing a specific ONOO- scavenger is remarkably important. In this study, the ability of hesperetin (3′,5,7-trihydroxy-4-methoxyflavanone) to scavenge ONOO- and to protect cells against ONOO- and ROS was investigated. The data gained show that hesperetin can efficiently scavenge authentic ONOO-. In spectrophotometric analysis, the data revealed that hesperetin led to declined ONOO--mediated nitration of tyrosine through electron donation. Hesperetin exhibited significant inhibition on the nitration of bovine serum albumin (BSA) by ONOO- in a dose-dependent manner. Hesperetin also manifested cytoprotection from cell damage induced by ONOO- and ROS. The present study suggests that hesperetin is a powerful ONOO- scavenger and promotes cellular defense activity in the protection against ONOO- involved diseases.

Inhibitory effects of water-soluble cationic manganese porphyrins on peroxynitrite-induced SOS response in Salmonella typhimurium TA4107/pSK1002

We have investigated the protective effects of water-soluble cationic Mn(III) porphyrins against peroxynitrite (ONOO −)-induced DNA damage in the cells of Salmonella typhimurium TA4107/pSK1002 and lipid peroxidation of red blood cell membranes. Mn(III) tetrakis ( N-methylpyridinium-4-yl) porphine (TMPyP) and the brominated form, Mn(III) octabromo-tetrakis ( N-methylpyridinium-4-yl) porphine (OBTMPyP) effectively reduced the damage and peroxidation induced by N-morpholino sydnonimine (SIN-1), which gradually generates ONOO − from O 2 − and NO produced through hydrolysis. Mn(III)OBTMPyP became 10-fold more active than the non-brominated form. In the presence of authentic ONOO −, the Mn(III) porphyrins were ineffective against damage and strongly enhanced lipid peroxidation, while the coexistence of ascorbic acid inhibited peroxidation. Using a diode array spectrophotometry, the reactions of Mn(III)TMPyP with authentic ONOO − and SIN-1 were measured. Mn(III)TMPyP is known to be catalytic for ONOO − decomposition in the presence of antioxidants. OxoMn(IV)TMPyP with SIN-1 was rapidly reduced back to Mn(III) without adding any oxidants. Further, in the SIN-1 system, the concentration of NO 2 − and NO 3 − were colorimetrically determined by Griess reaction based on the two-step diazotization. NO 2 − increased by addition of Mn(III) porphyrin and the ratio of NO 2 − to NO 3 − was 4–7 times higher than that (1.05) of SIN-1 alone. This result suggests that O 2 − from SIN-1 acts as a reductant and NO cogenerated is oxidized to NO 2 −, a primarily decomposition product of NO. Under the pathological conditions where biological antioxidants are depleted and ONOO − and O 2 − are extensively generated, the Mn(III) porphyrins will effectively cycle ONOO − decomposition using O 2 −.

Peroxynitrite-scavenging constituents from the brown algaSargassum thunbergii

Peroxynitrite formationin vivo is implicated in numerous human diseases and there is considerable interest in the use of antioxidants and natural products for their treatment. The three components (1–3) isolated fromSargassum thunbergii as well as the organic solventsoluble fractions and the aqueous layer ofS. thunbergii were evaluated for their potential to scavenge authentic ONOO and ONOO derived from 3-morpholinosydnonimine (SIN-1). The antioxidant activity of the individual fractions was in the order of 85% aquaous (aq.) MeOH>n-BuOH>n-hexane> H2O. The three known compounds sargahydroquinoic acid (1), sargaquinoic acid (2) and sargachromenol (3) showed peroxynitrite-scavenging activities comparable to those of L-ascorbic acid and penicillamine. These results showed a possible antioxidant activity in major constituents ofS. thunbergii.

Protecting Against Peroxynitrite-Mediated Cytotoxicity in Vascular Smooth Muscle Cells Via Upregulating Endogenous Glutathione Biosynthesis by 3H-1,2-dithiole-3-thione

Peroxynitrite (ONOO(-)) is critically involved in the pathogenesis of cardiovascular diseases. Reaction with glutathione (GSH) was proposed to be a major detoxification pathway of ONOO(-) in the biological system. This study was undertaken to determine if chemically elevated intracellular GSH affords protection against ONOO(-)-mediated toxicity in vascular cells. Incubation of aortic smooth muscle A10 cells with 3H-1,2-dithiole-3-thione (D3T) led to a concentration- and time-dependent elevation of cellular GSH. Treatment of the cells with D3T also augmented protein and gene expression of gamma-glutamylcysteine ligase. To examine the effects of D3T-induced GSH on ONOO(-)-mediated toxicity, we pretreated A10 cells with D3T and then exposed them to either authentic ONOO(-) or the ONOO(-) generator, 3-morpholinosydnonimine. We observed that D3T pretreatment of A10 cells resulted in a significant protection against ONOO(-) cytotoxicity. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of ONOO(-) cytotoxicity. To further demonstrate the causal involvement of GSH induction in D3T cytoprotection, we cotreated A10 cells with BSO to abolish D3T-induced GSH elevation. BSO cotreatment was found to greatly reverse the protective effects of D3T on ONOO(-)-elicited cytotoxicity. Taken together, our results demonstrate that upregulating GSH biosynthesis by D3T results in a marked protection against ONOO(-)-induced toxicity in vascular cells.

Peroxynitrite inhibits Ca2+-activated K+ channel activity in smooth muscle of human coronary arterioles.

We examined the hypothesis that ONOO-, a product of the interaction between superoxide (O2*-) and nitric oxide (NO), inhibits calcium-activated K+ (KCa) channel activity in vascular smooth muscle cells (VSMCs) of human coronary arterioles (HCAs), thereby reducing hyperpolarization-mediated vasodilation. HCAs were dissected from right atrial appendages. The interaction of ONOO- with microvessels was determined by immunohistochemistry using a nitrotyrosine antibody. Strong staining was observed in arteries exposed to authentic ONOO- or to sodium nitroprusside (SNP)+xanthine (XA)+xanthine oxidase (XO). Dilation to 10(-8) mol/L bradykinin (BK) was abolished in vessels exposed to ONOO- (-2.5+/-8%; P<0.05) but not DC-ONOO- (65+/-8%). Reduced dilation to BK was also observed after application of XO and SNP. Dilation to NS1619 (KCa channel opener) was reduced in endothelial denuded arterioles treated with ONOO-. In isolated VSMCs, whole-cell peak K+ current density was reduced by ONOO- (control 65+/-15 pA/pF; ONOO- 42+/-9 pA/pF; P<0.05). Iberiotoxin had no further effect on whole-cell K+ current. In inside-out patches, ONOO- but not DC-ONOO- decreased open state probability (NP(o)) of KCa channel by 50+/-12%. O2*- generated by XA+XO had no effect on BK-induced dilation and NP(o) of KCa channels. These results suggest that ONOO-, but not O2*-, inhibits KCa channel activity in VSMCs possibly by a direct effect. This mechanism may contribute to impaired EDHF-mediated dilation in conditions such as ischemia/reperfusion where increased activity of NO synthase occurs in the presence of excess of O2*-.

Peroxynitrite scavenging activity of herb extracts.

Peroxynitrite (ONOO(-)) is a cytotoxicant with strong oxidizing properties toward various cellular constituents, including sulphydryls, lipids, amino acids and nucleotides and can cause cell death, lipid peroxidation, carcinogenesis and aging. The aim of this study was to characterize ONOO(-) scavenging constituents from herbs. Twenty-eight herbs were screened for their ONOO(-) scavenging activities with the use of a fluorometric method. The potency of scavenging activity following the addition of authentic ONOO(-) was in the following order: witch hazel bark > rosemary > jasmine tea > sage > slippery elm > black walnut leaf > Queen Anne's lace > Linden flower. The extracts exhibited dose-dependent ONOO(-) scavenging activities. We found that witch hazel (Hamamelis virginiana L.) bark showed the strongest effect for scavenging ONOO(-) of the 28 herbs. Hamamelitannin, the major active component of witch hazel bark, was shown to have a strong ability to scavenge ONOO(-). It is suggested that hamamelitannin might be developed as an effective peroxynitrite scavenger for the prevention of ONOO(-) involved diseases.

Enhanced vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteinases.

Peroxynitrite (ONOO ‐), which is generated from nitric oxide (NO) and superoxide anion (O2‐) under pathological conditions, plays an important role in pathophysiological processes. Activation of matrix metalloproteinases (MMPs) contributes to tumor angiogenesis and metastasis. NO mediates the enhanced vascular permeability and retention (EPR) effect in solid tumors, and ONOO‐ activates proMMP to MMP in vitro. In this study, we examined the role of ONOO‐ in the EPR effect in solid tumors and normal tissues as related to MMP activation. Authentic ONOO‐, at 50 nmol or higher concentrations, induced the enhanced vascular permeability in normal dorsal skin of mice. ONOO‐ scavengers ebselen and uric acid significantly suppressed the EPR effect in mouse sarcoma 180 (S‐180) tumors. Indirect evidence for formation of ONOO‐ in S‐180 and mouse colon adenocarcinoma (C‐38) tumors included strong immunostaining for nitrotyrosine in the tumor tissue, predominantly surrounding the tumor vessels. MMP inhibitor BE16627B (66.6 mg/kg i.v., given 2 tunes) or SI‐27 (10 mg/kg i.p., given 2 times) significantly suppressed the ONOO‐induced EPR effect in S‐180 tumors and in normal skin. Soybean trypsin inhibitor (Kunitz type), broad‐spectrum proteinase inhibitor ovomacroglobulin, and bradykinin receptor antagonist HOE 140 also significantly suppressed the ONOO‐induced EPR effect in normal skin tissues. These data suggest that ONOO‐ may be involved in and promote the EPR effect in tumors, which could be mediated partly through activation of MMPs and a subsequent proteinase cascade to generate potent vasoactive mediators such as bradykinin.

Glutathione reverses endothelial damage from peroxynitrite, the byproduct of nitric oxide degradation, in crystalloid cardioplegia.

NO has been advocated as an adjunct to cardioplegia solutions. However, NO undergoes a rapid biradical reaction with superoxide anions to produce peroxynitrite (ONOO(-)). ONOO(-) in crystalloid cardioplegia solution induces injury to coronary endothelium and to systolic function after cardioplegia and reperfusion. However, ONOO(-) may be degraded to less lethal or cardioprotective intermediates with glutathione (GSH) in reactions separate from its well known antioxidant effects. We hypothesized that GSH detoxifies ONOO(-) and reverses defects in endothelial function and systolic function when present in crystalloid cardioplegia. In anesthetized dogs on cardiopulmonary bypass, a 45-minute period of global normothermic ischemia was followed by 60 minutes of intermittent cold crystalloid cardioplegia (Plegisol) and 2 hours of reperfusion. The cardioplegia solution contained 5 micromol/L authentic ONOO(-); catalase was included to attenuate the potential antioxidant effects of GSH and to unmask the effects on ONOO(-). In 1 group (CP+GSH, n=5), the cardioplegia contained 500 micromol/L GSH, whereas 1 group received crystalloid cardioplegia without GSH (CCP, n=6). There were no group differences in postcardioplegia left ventricular systolic function (end-systolic pressure-volume relation, impedance catheter: CCP 10.0+/-2.4 versus CP+GSH 10.6+/-1.3 mm Hg/mL) or diastolic chamber stiffness (ss-coefficient: CCP 0.35+/-0.2 versus CP+GSH 0.31+/-0.18). Myocardial neutrophil accumulation (myeloperoxidase activity) was attenuated in CP+GSH versus CCP (2.2+/-0.7 versus 5.4+/-1.2, P:<0.05). In postexperimental coronary arteries, maximal endothelium-dependent relaxation was greater in CP+GSH than in CCP (118+/-6% versus 92+/-5%, P:<0.05), with a smaller EC(50) value (-7. 10+/-0.05 versus -6.98+/-0.03, respectively, P:<0.05). Smooth muscle relaxation was complete in both groups. The adherence of neutrophils to postexperimental coronary arteries as a measure of endothelial function was less in CP+GSH than in CCP (98+/-18 versus 234+/-36 neutrophils/mm(2), P:<0.05). Nitrosoglutathione, a byproduct of the reaction between ONOO(-) and GSH, was greater in CP+GSH than in CCP (4.1+/-2.3 versus 0.4+/-0.2 microg/mL, P:<0.05). GSH in crystalloid cardioplegia detoxifies ONOO(-) and forms cardioprotective nitrosoglutathione, resulting in attenuated neutrophil adherence and selective endothelial protection through the inhibition of neutrophil-mediated damage.

Glutathione Reverses Endothelial Damage From Peroxynitrite, the Byproduct of Nitric Oxide Degradation, in Crystalloid Cardioplegia

Background —NO has been advocated as an adjunct to cardioplegia solutions. However, NO undergoes a rapid biradical reaction with superoxide anions to produce peroxynitrite (ONOO − ). ONOO − in crystalloid cardioplegia solution induces injury to coronary endothelium and to systolic function after cardioplegia and reperfusion. However, ONOO − may be degraded to less lethal or cardioprotective intermediates with glutathione (GSH) in reactions separate from its well known antioxidant effects. We hypothesized that GSH detoxifies ONOO − and reverses defects in endothelial function and systolic function when present in crystalloid cardioplegia. Methods and Results —In anesthetized dogs on cardiopulmonary bypass, a 45-minute period of global normothermic ischemia was followed by 60 minutes of intermittent cold crystalloid cardioplegia (Plegisol) and 2 hours of reperfusion. The cardioplegia solution contained 5 μmol/L authentic ONOO − ; catalase was included to attenuate the potential antioxidant effects of GSH and to unmask the effects on ONOO − . In 1 group (CP+GSH, n=5), the cardioplegia contained 500 μmol/L GSH, whereas 1 group received crystalloid cardioplegia without GSH (CCP, n=6). There were no group differences in postcardioplegia left ventricular systolic function (end-systolic pressure-volume relation, impedance catheter: CCP 10.0±2.4 versus CP+GSH 10.6±1.3 mm Hg/mL) or diastolic chamber stiffness (β-coefficient: CCP 0.35±0.2 versus CP+GSH 0.31±0.18). Myocardial neutrophil accumulation (myeloperoxidase activity) was attenuated in CP+GSH versus CCP (2.2±0.7 versus 5.4±1.2, P &lt;0.05). In postexperimental coronary arteries, maximal endothelium-dependent relaxation was greater in CP+GSH than in CCP (118±6% versus 92±5%, P &lt;0.05), with a smaller EC 50 value (−7.10±0.05 versus −6.98±0.03, respectively, P &lt;0.05). Smooth muscle relaxation was complete in both groups. The adherence of neutrophils to postexperimental coronary arteries as a measure of endothelial function was less in CP+GSH than in CCP (98±18 versus 234±36 neutrophils/mm 2 , P &lt;0.05). Nitrosoglutathione, a byproduct of the reaction between ONOO − and GSH, was greater in CP+GSH than in CCP (4.1±2.3 versus 0.4±0.2 μg/mL, P &lt;0.05). Conclusions —GSH in crystalloid cardioplegia detoxifies ONOO − and forms cardioprotective nitrosoglutathione, resulting in attenuated neutrophil adherence and selective endothelial protection through the inhibition of neutrophil-mediated damage.