Nitration of annexin II tetramer.

Annexin II tetramer (AII(t)) is a member of the Ca(2+)- and phospholipid-binding protein family and is implicated in membrane fusion during surfactant secretion. It had previously been shown that high concentrations of nitric oxide (NO) inhibit surfactant secretion from lung type II cells. NO reacts with superoxide (O(2)(-)) to form peroxynitrite (ONOO(-)), a tyrosine nitrating agent, which is found in lungs under certain pathological conditions. It is therefore hypothesized that nitration of AII(t) by ONOO(-) may be a mechanism for the NO inhibition of regulated exocytosis. We therefore performed in vitro studies to test effects of ONOO(-) on AII(t). Western blot analysis using anti-nitrotyrosine antibodies showed a dose-dependent nitration of tyrosine residues in AII(t) treated with ONOO(-). Nitration occurred on the core domain of the p36 subunit, as well as on the p11 subunit. ONOO(-) also caused the formation of dimers between p36 and p11 subunits which were stable in the presence of heating, SDS, and beta-mercaptoethanol. AII(t)-mediated liposome aggregation was inhibited by ONOO(-) with an IC(50) of approximately 30 microM. The inhibition was abolished by urate (a scavenger of ONOO(-) and *OH), but not by mannitol (a scavenger of *OH) or superoxide dismutase (a scavenger of O(2)(-)) and appeared to be specific to AII(t), since ONOO(-) only slightly influenced annexin I-mediated liposome aggregation. The conformational change of AII(t) induced by Ca(2+) had no effect on the inhibition. Furthermore, ONOO(-) only partially inhibited the binding of AII(t) to membranes. Nitration of AII(t) also occurred in intact A549 cells, a lung epithelial cell line, treated with ONOO(-). The results of this study suggest that AII(t)-mediated liposome aggregation was inhibited by nitration of the protein.     

Oxidation of 5-thio-2-nitrobenzoic acid, by the biologically relevant oxidants peroxynitrite anion, hydrogen peroxide and hypochlorous acid.

The modification of protein and non-protein thiols by oxidants including hydrogen peroxide (H(2)O(2)), peroxynitrite anion (ONOO(-)) and hypochlorous acid (HOCl) is well documented. Using an aromatic thiol, 5-thio-2-nitrobenzoic acid, and biologically relevant oxidants, we have identified higher oxidation states of sulfur including the sulfonic acid derivative and the disulfide S-oxide, a thiosulfinate, by HPLC and mass spectrometry. The initial reaction of ONOO(-) with 5-thio-2-nitrobenzoic acid yielded a transient red intermediate, the sulfenate anion. The red intermediate was observed when ONOO(-) and H(2)O(2) were used to oxidize 5-thio-2-nitrobenzoic acid and it persisted for several seconds at pH 7. HOCl oxidized the disulfide, 5,5'dithiobis(2-nitrobenzoic acid) to the corresponding sulfonic acid and no additional products were detected. Using this system, we can directly compare the thiol-oxidizing abilities of several oxidants. Because 5-thio-2-nitrobenzoic acid is the product of the reaction of Ellman's reagent with protein thiols, a detailed study of its stability in biological matrices where oxidants may be generated is warranted.     

Hypochlorous acid is a potent inhibitor of GST P1-1.

Glutathione S-transferase is a phase II detoxification enzyme that can be inactivated by H(2)O(2). During oxidative stress various other reactive oxygen species are generated that are more reactive than the relatively stable H(2)O(2). Hypochlorous acid (HOCl) is a powerful oxidant which is highly reactive towards a range of biological substrates. We studied the influence of HOCl on the activity of GST P1-1. HOCl inhibits purified glutathione S-transferase P1-1 in a concentration dependent manner with an IC(50)-value of 0.6 microM, which is more than 1000 times as low as IC(50) reported for H(2)O(2). HOCl lowered the V(max) value, but did not affect the K(m) for CDNB. Our results show that HOCl is a potent, non-competitive inhibitor of GST P1-1. The relevance of this effect is discussed.     

Potential role of methionine sulfoxide in the inactivation of the chaperone GroEL by hypochlorous acid (HOCl) and peroxynitrite (ONOO-)

GroEL is an Escherichia coli molecular chaperone that functions in vivo to fold newly synthesized polypeptides as well as to bind and refold denatured proteins during stress. This protein is a suitable model for its eukaryotic homolog, heat shock protein 60 (Hsp60), due to the high number of conserved amino acid sequences and similar function. Here, we will provide evidence that GroEL is rather insensitive to oxidants produced endogenously during metabolism, such as nitric oxide (.NO) or hydrogen peroxide (H(2)O(2)), but is modified and inactivated by efficiently reactive species generated by phagocytes, such as peroxynitrite (ONOO(-)) and hypochlorous acid (HOCl). For the exposure of 17.5 microm GroEL to 100-250 microm HOCl, the major pathway of inactivation was through the oxidation of methionine to methionine sulfoxide, established through mass spectrometric detection of methionine sulfoxide and the reactivation of a significant fraction of inactivated GroEL by the enzyme methionine sulfoxide reductase B/A (MsrB/A). In addition to the oxidation of methionine, HOCl caused the conversion of cysteine to cysteic acid and this product may account for the remainder of inactivated GroEL not recoverable through MsrB/A. In contrast, HOCl produced only negligible yields of 3-chlorotyrosine. A remarkable finding was the conversion of Met(111) and Met(114) to Met sulfone, which suggests a rather low reduction potential of these 2 residues in GroEL. The high sensitivity of GroEL toward HOCl and ONOO(-) suggests that this protein may be a target for bacterial killing by phagocytes.     

Uric acid, a peroxynitrite scavenger, inhibits CNS inflammation, blood-CNS barrier permeability changes, and tissue damage in a mouse model of multiple sclerosis.

Peroxynitrite (ONOO(-)), a toxic product of the free radicals nitric oxide and superoxide, has been implicated in the pathogenesis of CNS inflammatory diseases, including multiple sclerosis and its animal correlate experimental autoimmune encephalomyelitis (EAE). In this study we have assessed the mode of action of uric acid (UA), a purine metabolite and ONOO(-) scavenger, in the treatment of EAE. We show that if administered to mice before the onset of clinical EAE, UA interferes with the invasion of inflammatory cells into the CNS and prevents development of the disease. In mice with active EAE, exogenously administered UA penetrates the already compromised blood-CNS barrier, blocks ONOO(-)-mediated tyrosine nitration and apoptotic cell death in areas of inflammation in spinal cord tissues and promotes recovery of the animals. Moreover, UA treatment suppresses the enhanced blood-CNS barrier permeability characteristic of EAE. We postulate that UA acts at two levels in EAE: 1) by protecting the integrity of the blood-CNS barrier from ONOO(-)-induced permeability changes such that cell invasion and the resulting pathology is minimized; and 2) through a compromised blood-CNS barrier, by scavenging the ONOO(-) directly responsible for CNS tissue damage and death.     

Enzymatic nitration of phytophenolics: evidence for peroxynitrite-independent nitration of plant secondary metabolites

Peroxynitrite (ONOO(-)), a reactive nitrogen species, is capable of nitrating tyrosine residue of proteins. Here we show in vitro evidence that plant phenolic compounds can also be nitrated by an ONOO(-)-independent mechanism. In the presence of NaNO(2), H(2)O(2), and horseradish peroxidase (HRP), monophenolic p-coumaric acid (p-CA, 4-hydroxycinnamic acid) was nitrated to form 4-hydroxy-3-nitrocinnamic acid. The reaction was completely inhibited by KCN, an inhibitor for HRP. The antioxidant ascorbate suppressed p-CA nitration and its suppression time depended strongly on ascorbate concentration. We conclude that nitrogen dioxide radical (NO(2)(radical)), but not ONOO(-), produced by a guaiacol peroxidase is the intermediate for phytophenolic nitration.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Reactive oxygen species and oocyte aging: role of superoxide, hydrogen peroxide, and hypochlorous acid

Aging of the unfertilized oocyte inevitably occurs following ovulation, limiting its fertilizable life span. However, the mechanisms that regulate oocyte aging are still unclear. We hypothesize that reactive oxygen species such as superoxide (O2-), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are likely candidates that may initiate these changes in the oocyte. In order to test this hypothesis, we investigated direct effects of O2- [hypoxanthine/xanthine oxidase system generating 0.12 (n=42) and 0.25 (n=45) microM O2-/min], H2O2 (20 or 100 microM, n=60), and HOCl, (1, 10, and 100 microM, n=50) on freshly ovulated or relatively old mouse oocytes, while their sibling oocytes were fixed immediately or cultured under physiological conditions (n=96). The aging process was assessed by the zona pellucida dissolution time (ZPDT), ooplasm microtubule dynamics (OMD), and cortical granule (CG) status. The ZPDT increased 2-fold in relatively old, compared to young, untreated oocytes (P<0.0001). Exposure to O2- increased it even further (P<0.0001). Similarly, more O2- exposed oocytes exhibited increased OMD and major CG loss, with fewer having normal OMD and intact CG compared to untreated controls. Interestingly, young oocytes resisted "aging," when exposed to 20 microM H2O2, while the same enhanced the aging phenomena in relatively old oocytes (P<0.05). Exposure to even very low levels of HOCl induced the aging phenomena in young and relatively old oocytes, and higher concentrations of HOCl compromised oocyte viability. Overall, O2-, H2O2, and HOCl each augment oocyte aging, more so in relatively old oocytes, suggesting compromised antioxidant capacity in aging oocytes.     

Peroxynitrite participates in mechanisms involved in capacitation of cryopreserved cattle

The effect of peroxynitrite (ONOO(-)) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin (10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1-20 microM), a ONOO(-) donor. The participation of ONOO(-) was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2-20 mM). Spermatozoa capacitated with SIN-1 were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO(-) during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO(-) was evaluated by incubation with specific inhibitors (50 microM H-89, 0.1 microM bisindolylmaleimide I, and 3 microM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 microM SIN-1 treatment (23+/-2%) were significantly greater with respect to the control (4.6+/-1.62%). At 15 min of incubation the greatest capacitation was observed (P<0.05), reaching a plateau between 15 and 45 min. Follicular fluid induced acrosome reaction in spermatozoa previously capacitated with 10 microM SIN-1 (P<0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify the capacitation induced by SIN-1 (27.4+/-3.85 and 24.8+/-4.75, respectively). Genistein, a PTK inhibitor, produced a significant capacitation decrease (8.6+/-5.5%). These results indicate that endogenous ONOO(-) may be generated during heparin- or SNP-induced capacitation. Exogenous ONOO(-) acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.     

Endothelium-dependent hypotensive and vasorelaxant effects of the essential oil from aerial parts of Mentha x villosa in rats.

Cardiovascular effects of an essential oil from the aerial parts of Mentha x villosa (OEMV) were tested in rats using a combined in vivo and in vitro approach. In non-anesthetized normotensive rats, OEMV (1, 5, 10, 20, 30 mg kg(-1) body wt., i.v.) induced a significant and dose-dependent hypotension (-3 +/- 1.8%; -6 +/- 0.7%; -40 +/- 6.7%; -58 +/- 3.8%; -57 +/- 2.1%, respectively) associated with decreases in heart rate (-1 +/- 0.3%; -9 +/- 0.9%; -17 +/- 3.2%; -72 +/- 3.1%; -82 +/- 1.4%, respectively). The hypotensive and bradycardic responses evoked by OEMV were attenuated and blocke by pre-treatment of the animals with atropine (2 mg kg(-1) body wt., i.v.). In isolated rat atrial preparations, OEMV (10, 100, 300, 500 microg ml(-1)) produced concentration-related negative chronotropic and inotropic effects (IC50 value = 229 +/- 17 and 120 +/- 13 microg ml(-1), respectively). In isolated rat aortic rings, increasing concentrations of OEM (10, 100, 300, 500 microg ml(-1)) were able to antagonize the effects of phenylephrine (1 microM), prostaglandin F2alpha (10 microM) and KCl (80 mM)-induced contractions (IC50 value = 255 +/- 9, 174 +/- 4 and 165 +/- 14 microg ml(-1), respectively). The vasorelaxant activity induced by OEMV was attenuated significantly by either endothelium removal (IC50 value = 304 +/- 9 microg ml(-1)), NG-nitro L-arginine methyl ester (L-NAME) 100 microM (IC50 value=359 +/- 18 microg ml(-1)), L-NAME 300 microM (IC50 value = 488 +/- 20 microg ml(-1)) or indomethacin 10 microM (IC50 value = 334 +/- 18 microg ml(-1)). However, it was not affected by atropine 1 microM (IC50 value = 247 +/- 12 microg ml(-1)). Furthermore, the hypotensive response induced by OEMV was attenuated significantly after nitric oxide (NO) synthase blockade (L-NAME, 20 mg kg(-1) body wt., i.v.), while bradycardia was not altered. The results suggest that the hypotensive effect induced by OEMV is probably due to its direct cardiodepressant action and peripheral vasodilation, which can be attributed to both endothelium-dependent (via EDRFs, at least NO and prostacyclin) and endothelium-independent mechanisms (such as Ca2+ channel blockade).     

Tyrosine nitration by peroxynitrite formed from nitric oxide and superoxide generated by xanthine oxidase

Peroxynitrite (ONOO(-)) is a potent nitrating and oxidizing agent that is formed by a rapid reaction of nitric oxide (NO) with superoxide anion (O(2)). It appears to be involved in the pathophysiology of many inflammatory and neurodegenerative diseases. It has recently been reported (Pfeiffer, S., and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285) that ONOO(-) generated at neutral pH from NO and O(2) (NO/O(2)) was substantially less efficient than preformed ONOO(-) at nitrating tyrosine. Here we re-evaluated tyrosine nitration by NO/O(2) with a shorter incubation period and a more sensitive electrochemical detection system. Appreciable amounts of nitrotyrosine were produced by ONOO(-) formed in situ (2.9 micrometer for 5 min; 10 nm/s) by NO/O(2) flux obtained from propylamine NONOate (CH(3)N[N(O)NO](-) (CH(2))(3)NH(2)(+)CH(3)) and xanthine oxidase using pterin as a substrate in phosphate buffer (pH 7.0) containing 0.1 mm l-tyrosine. The yield of nitrotyrosine by this NO/O(2) flux was approximately 70% of that produced by the same flux of preformed ONOO(-) (2.9 micrometer/5 min). When hypoxanthine was used as a substrate, tyrosine nitration by NO/O(2) was largely eliminated because of the inhibitory effect of uric acid produced during the oxidation of hypoxanthine. Tyrosine nitration caused by NO/O(2) was inhibited by the ONOO(-) scavenger ebselen and was enhanced 2-fold by NaHCO(3), as would be expected, because CO(2) promotes tyrosine nitration. The profile of nitrotyrosine and dityrosine formation produced by NO/O(2) flux (2.9 micrometer/5 min) was consistent with that produced by preformed ONOO(-). Tyrosine nitration predominated compared with dityrosine formation caused by a low nanomolar flux of ONOO(-) at physiological concentrations of free tyrosine (<0.5 mm). In conclusion, our results show that NO generated with O(2) nitrates tyrosine with a reactivity and efficacy similar to those of chemically synthesized ONOO(-), indicating that ONOO(-) can be a significant source of tyrosine nitration in physiological and pathological events in vivo.     

Human hemi-myeloperoxidase. Initial chlorinating activity at neutral pH, compound II and III formation, and stability towards hypochlorous acid and high temperature.

Human neutrophilic myeloperoxidase (MPO) is involved in the defence mechanism of the body against micro-organisms. The enzyme catalyses the generation of the strong oxidant hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. In normal neutrophils MPO is present in the dimeric form (140 kDa). The disulphide-linked protomers each consist of a heavy subunit and a light one. Reductive alkylation converts the dimeric enzyme into two promoters, 'hemi-myeloperoxidase'. We studied the initial activities of human dimeric MPO and hemi-MPO at the physiological pH of 7.2 and found no significant differences in chlorinating activity. These results indicate that, at least at neutral pH, the protomers of MPO function independently. The absorption spectra of MPO compounds II and III, both inactive forms concerning HOCl generation, and the rate constants of their formation were the same for dimeric MPO and hemi-MPO, but hemi-MPO required a slightly larger excess of H2O2 for complete conversion. Hemi-MPO was less stable at a high temperature (80 degrees C) as compared to the dimeric enzyme. Furthermore, the resistance of the chlorinating activity of hemi-MPO against its oxidative product hypochlorous acid was somewhat lower (IC50 = 32 microM HOCl) compared to dimeric MPO (IC50 = 50 microM HOCl). The higher stability of dimeric MPO in the presence of its oxidative product compared to that of monomeric MPO might be the reason for the occurrence of MPO as a dimer.     

Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides

Pedilanthus tithymaloides (L.) Poit. (Euphorbiaceae) is a low tropical American shrub with a reported wide range of healing properties such as emetic, anti-inflammatory, antibiotic, antiseptic, antihemorrhagic, antiviral, antitumoral, and abortive. In the present study, a tincture from P. tithymaloides collected in Cuba was evaluated for its in vivo anti-inflammatory activity, using the rat paw oedema assay, and for its in vitro scavenging effects on reactive oxygen species (ROS) (HO*, O2*-, HOCl, ROO* and H2O2), reactive nitrogen species (RNS) (ONOO- and *NO), and DPPH* radical. The protein, free amino acid, and phenolic contents of the tincture were also determined. Pertaining to the anti-inflammatory activity, the intraperitoneal administration of the tincture inhibited carrageenan-induced rat paw oedema, whereas in the scavenging assays the tincture showed to be effective against all the assayed ROS and RNS, specially for HO* (IC50 = 345+/-77 microg/mL), O2*- (IC50 = 143+/-7 microg/mL), HOCl (IC50 = 113+/-20 microg/mL), ONOO- (IC50 = 44+/-3 microg/mL), and *NO (IC50 = 54+/-4 microg/mL), but displayed weak activity in the DPPH* assay. The protein content of the tincture was 0.70%, and twenty free amino acids were identified and quantified. The content of total phenolics was 17.4+/-0.15 mg of gallic acid equivalents (GAE)/g dry material. These results provide scientific support for the empirical use of P. tithymaloides tincture as an anti-inflammatory medicine.     

Hypochlorous acid inhibits glutathione S-conjugate export from human erythrocytes

It was found that the hypochlorous acid (HOCl) inhibits the active efflux of glutathione S-conjugates, 2,4-dinitrophenyl-S-glutathione (DNP-SG, c(50%)=258+/-24 microM HOCl) and bimane-S-glutathione (B-SG, c(50%)=125+/-16 microM HOCl) from human erythrocytes, oxidises intracellular reduced glutathione (the ratio [HOCl]/[GSH](oxidized)=4) and inhibits basal as well as 2,4-dinitrophenol- (DNP) and 2,4-dinitrophenyl-S-glutathione (DNP-SG)-stimulated Mg(2+)-ATPase activities of erythrocyte membranes. Multidrug resistance-associated protein (MRP1) mediates the active export of glutathione S-conjugates in mammalian cells, including human erythrocytes. A direct impairment of erythrocyte membrane MRP by hypochlorous acid was shown by electrophoresis and immunoblotting (c(50%)=478+/-36 microM HOCl). The stoichiometry of the MRP/HOCl reaction was 1:1. These results demonstrate that MRP can be one of the cellular targets for the inflammatory mediator hypochlorous acid.     

Scavenging of peroxynitrite by phycocyanin and phycocyanobilin from Spirulina platensis: protection against oxidative damage to DNA.

Peroxynitrite (ONOO(-)) is known to inactivate important cellular targets and also mediate oxidative damage in DNA. The present study has demonstrated that phycocyanin, a biliprotein from spirulina platensis and its chromophore, phycocyanobilin (PCB), efficiently scavenge ONOO(-), a potent physiological inorganic toxin. Scavenging of ONOO(-) by phycocyanin and PCB was established by studying their interaction with ONOO(-) and quantified by using competition kinetics of pyrogallol red bleaching assay. The relative antioxidant ratio and IC(50) value clearly indicate that phycocyanin is a more efficient ONOO(-) scavenger than PCB. The present study has also shown that PCB significantly inhibits the ONOO(-)-mediated single-strand breaks in supercoiled plasmid DNA in a dose-dependent manner with an IC(50) value of 2.9 +/- 0.6 microM. These results suggest that phycocyanin, has the ability to inhibit the ONOO(-)-mediated deleterious biological effects and hence has the potential to be used as a therapeutic agent.     

Selective involvement of superoxide anion, but not downstream compounds hydrogen peroxide and peroxynitrite, in tumor necrosis factor-alpha-induced apoptosis of rat mesangial cells

Tumor necrosis factor-alpha (TNF-alpha) induces reactive oxygen species (ROS) that serve as second messengers for intracellular signaling. Currently, precise roles of individual ROS in the actions of TNF-alpha remain to be elucidated. In this report, we investigated the roles of superoxide anion (O-(2)), hydrogen peroxide (H(2)O(2)), and peroxynitrite (ONOO(-)) in TNF-alpha-triggered apoptosis of mesangial cells. Mesangial cells stimulated by TNF-alpha produced O-(2) and underwent apoptosis. The apoptosis was inhibited by transfection with manganese superoxide dismutase or treatment with a pharmacological scavenger of O-(2), Tiron. In contrast, although exogenous H(2)O(2) induced apoptosis, TNF-alpha-triggered apoptosis was not affected either by transfection with catalase cDNA or by treatment with catalase protein or glutathione ethyl ester. Similarly, although ONOO(-) precursor SIN-1 induced apoptosis, treatment with a scavenger of ONOO(-), uric acid, or an inhibitor of nitric oxide synthesis, N(G)-nitro-L-argininemethyl ester hydrochloride, did not affect the TNF-alpha-triggered apoptosis. Like TNF-alpha-induced apoptosis, treatment with a O-(2)-releasing agent, pyrogallol, induced typical apoptosis even in the concurrent presence of scavengers for H(2)O(2) and ONOO(-). These results suggested that, in mesangial cells, TNF-alpha induces apoptosis through selective ROS. O-(2), but not H(2)O(2) or ONOO(-), was identified as the crucial mediator for the TNF-alpha-initiated, apoptotic pathway.     

Cyclic AMP and cyclic GMP independent stimulation of ventricular calcium current by peroxynitrite donors in guinea pig myocytes

We investigated the potential involvement of peroxynitrite (ONOO(-)) in the modulation of calcium current (I(Ca)) in guinea pig ventricular myocytes with the whole-cell patch clamp technique and with cyclic AMP (cAMP) measurements. Because of the short half-life of ONOO(-) at physiological pH, we induced an increase in its intracellular levels by using donors of the precursors, nitric oxide (NO) and superoxide anion (O(2) (-)). High concentrations of NO donors, SpermineNONOate (sp/NO, 300 microM) or SNAP (300 microM) increased basal I(Ca) (50.3 +/- 4.6%, n = 7 and 46.2 +/- 5.0%, n = 13). The superoxide anion donor Pyrogallol (100 microM) also stimulated basal I(Ca) (44.6 +/- 2.8%, n = 11). At lower concentration sp/NO (10 nM) and Pyrogallol (1 microM), although separately ineffective on I(Ca), enhanced the current if applied together (33.5 +/- 0.7%, n = 7). The simultaneous donor of O(2) (-) and NO, SIN-1 (500 microM), also stimulated basal I(Ca) (22.8 +/- 2.1%, n = 13). In the presence of saturating cyclic GMP (cGMP, 50 microM) in the patch pipette or of extracellular dibutyryl cGMP (dbcGMP, 100 microM), I(Ca) was still increased by SIN-1 (32.0 +/- 6.1%, n = 4 and 30.0 +/- 5.4%, n = 8). Both Manganese(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP, 100 microM) a ONOO(-) scavenger, and superoxide dismutase (SOD) (150 U/ml) reversed the stimulatory effect of SIN-1 on I(Ca) (respectively -0.6 +/- 4.1%, n = 4 and 3.6 +/- 4.3%, n = 4). Intracellular cAMP level was unaltered by SIN-1, while it was enhanced by blocking the NO-cGMP pathway with the NO synthase inhibitor L-NMMA. These results suggest that peroxynitrite donors increase cardiac calcium current without the involvement of cAMP and cGMP.     

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.     

Hemodynamics influences vascular peroxynitrite formation: Implication for low-density lipoprotein apo-B-100 nitration

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherogenesis. Superoxide anion (O2(-.)) reacts with nitric oxide (.NO) at a rapid diffusion-limited rate to form peroxynitrite (O2(-.) + .NO-->ONOO(-)). Immunohistostaining of human coronary arterial bifurcations or curvatures, where OSS develops, revealed the presence of nitrotyrosine staining, a fingerprint of peroxynitrite; whereas in straight segments, where PSS occurs, nitrotyrosine was absent. We examined vascular nitrative stress in models of oscillatory (OSS) and pulsatile shear stress (PSS). Bovine aortic endothelial cells (BAEC) were exposed to fluid shear stress that simulates arterial blood flow: (1) PSS at a mean shear stress (tau(ave)) of 23 dyn cm(-2) and a temporal gradient (partial differential(tau)/partial differential(t)) at 71 dyn cm(-2) s(-1), and (2) OSS at tau(ave) = 0.02 dyn cm(- 2) and partial differential(tau)/partial differential(t) = +/- 3.0 dyn cm(-2) s(-1) at a frequency of 1 Hz. OSS significantly up-regulated one of the NADPH oxidase subunits (NOx4) expression accompanied with an increase in O2(-.) production. In contrast, PSS up-regulated eNOS expression accompanied with .NO production (total NO(2)(-) and NO(3)(-)). To demonstrate that O2(-.) and .NO are implicated in ONOO(-) formation, we added low-density lipoprotein cholesterol (LDL) to the medium in which BAEC were exposed to the above flow conditions. The medium was analyzed for LDL apo-B-100 nitrotyrosine by liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS). OSS induced higher levels of 3-nitrotyrosine, dityrosine, and o-hydroxyphenylalanine compared with PSS. In the presence of ONOO(-), specific apo-B-100 tyrosine residues underwent nitration in the alpha and beta helices: alpha-1 (Tyr(144)), alpha-2 (Tyr(2524)), beta-2 (Tyr(3295)), alpha-3 (Tyr(4116)), and beta-2 (Tyr(4211)). Hence, the characteristics of shear stress in the arterial bifurcations influenced the relative production of O2(-.) and .NO with an implication for ONOO(-) formation as evidenced by LDL protein nitration.     

Biochemical characterization of some pyrazolopyrimidine-based inhibitors of xanthine oxidase

Inhibition of xanthine oxidase-catalyzed conversion of xanthine to uric acid by various pyrazolopyrimidine-based inhibitors (allopurinol derivatives) was evaluated and compared with the standard inhibitor allopurinol. Three compounds out of the seven compounds used in the study were found to be reasonably good inhibitors of xanthine oxidase (XO). 4-Amino-6-mercaptopyrazolo-3,4-d-pyrimidine was found to be the most potent inhibitor of XO (IC50 = 0.600 +/- 0.009 microM). 4-Mercapto-1H-pyrazolo-3,4-d-pyrimidine (IC50 = 1.326 +/- 0.013 microM) and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine (IC50 = 1.564 +/- 0.065 microM) also showed comparable inhibitory activity to that of allopurinol (IC50 = 0.776 +/- 0.012 microM). All three compounds showed competitive type of inhibition with comparable Ki values. Induction of the electron transfer reaction catalyzed by XO in the presence of these compounds monitored as reduction of 2,6-dichlorophenolindophenol (DCPIP) revealed that electron transfer by 4-amino-6-mercaptopyrazolo-3,4-d-pyrimidine is comparable to that obtained by allopurinol or xanthine. However, 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine and 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine did not show DCPIP reduction. On the other hand, enzymatic reduction of cytochrome c in the presence of the three compounds was found to be insignificant and much less in comparison to allopurinol and xanthine. Therefore, both 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine and 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine displayed the inhibitory property and also did not produce XO-mediated reactive oxygen species (ROS). Since 4-mercapto-1H-pyrazolo-3,4-d-pyrimidine was found to have some toxicity, the effect of 4-amino-6-hydroxypyrazolo-3,4-d-pyrimidine on the enzymatic formation of uric acid and ROS was investigated and it was found that this compound was inhibiting enzymatic generation of both uric acid and ROS. It can be noted that the standard inhibitor, allopurinol, inhibits uric acid formation but produces ROS.