Nitric Oxide-Mediated Augmentation of Neutrophil Reactive Oxygen and Nitrogen Species Formation: Critical Use of Probes (original) (raw)
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British Journal of Pharmacology, 1996
The effects of oxygen free radical scavengers and endothelial cell-derived nitric oxide (EDNO) on the death of porcine cultured aortic endothelial cells exposed to exogenous superoxide-[xanthine (0.4 mM)/ xanthine oxidase (0.04 unit ml-')+ diethylenetriaminepentaacetic acid (DTPA, 10 uM)] or hydroxyl radical-generating system(s) [superoxide generating system+ ferric iron (Fe3, 0.1 mM) or peroxynitrite (0-100 uM)] have been evaluated. 2 Spin trapping studies using 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) with electron paramagnetic resonance spectrometry were also conducted to determine qualitatively the oxidant species generated by the oxidant generating systems. 3 Endothelial cell injury provoked by the exogenous superoxide generating system was inhibited by catalase, DTPA and a hydroxyl radical scavenger (dimethyl sulphoxide, DMSO), but not by superoxide dismutase (SOD). Addition of Fe3+ to the superoxide generating system enhanced the cell injury. These suggested that the direct cytotoxicity of exogenous superoxide is limited, and that endogenous transition metal-dependent hydroxyl radical formation is involved in the cell injury. 4 An inhibitor of the constitutive NO-pathway, NG-monomethyl-L-arginine, did not influence cell injury induced by the superoxide generating system, suggesting that basal NO production is not responsible for the cytotoxicity. 5 Stimulation of endothelial cells with bradykinin enhanced cell injury provoked by the exogenous superoxide generating system, but not by the exogenous hydroxyl radical generating system. The enhancement by bradykinin was inhibited by NG-monomethyl-L-arginine and bradykinin B2-receptor antagonist, D-Arg-[Hyp3, Thi5'8, D-Phe7] bradykinin, suggesting that an interaction of NO with superoxide is involved in the enhanced cytotoxicity. A possible intermediate of this reaction, peroxynitrite, also caused endothelial cell injury in a concentration-dependent manner. 6 The modulatory effects of NO on hydroxyl radical-like activity (=formaldehyde production) from the superoxide generating system was also evaluated in a cell-free superoxide/NO generating system, consisting of xanthine/xanthine oxidase, DTPA, DMSO, and various amounts of a spontaneous NO generator, sodium nitroprusside (SNP) and were compared with those of Fe3". At doses up to 10 gM, SNP concentration-dependently increased the formaldehyde production while the higher concentrations of SNP decreased. The maximum amount of formaldehyde produced by SNP was 5 fold less than that produced by Fe3+ (0.1 mM). Peroxynitrite-induced formaldehyde formation was concentrationdependently inhibited by SNP. 7 We conclude that agonist-stimulated but not basal NO production acts as cytotoxic hydroxyl radical donor as well as the endogenous transition metal when endothelial cells are exposed to exogenous superoxide anion, while the modulatory effect of EDNO is limited by a secondary reaction with hydroxyl radicals.
Biochemical and Biophysical Research Communications, 1991
The oxygenderived free radical superoxide anion (.Oz') plays an important role in the pathogenesis of various diseases. Recent demonstrations that .Ozs inactivates the potent vasodilator endotheliumderived relaxing factor (EDRF) and that EDRF is probably nitric oxide (NO) suggest that EDRF(N0) may act as an endogenous free radical scavenger. This hypothesis was tested in an in vitro system by analyzing the effect of authentic NO (dilutions of a saturated aqueous solution) on .Oz' production (detected spectrophotometrically as reduction of cytochrome @ by fMet-Leu-Phe-activated human leukocytes (PMN). NO depressed the rate of reduction of cytochrome g by ,Oz' released from PMN's or generated from the oxidation of hypoxanthine by xanthine oxkfase. This effect was concentration-dependent andoccurred at dilutions of the saturated NO solution (1:250 to 1 :lO) which inhibited platelet aggregation. NO had no direct effect on cytochrome g or on xanthine oxidase. These observations indicate that NO(EDRF) can be regarded as a scavenger of superoxide anion and they suggest that EDRF(N0) may provide a chemical barrier to cytotoxic free radicals u&-l.
Journal of Leukocyte Biology, 1997
Because nitric oxide (NO) can act both as a regulatory and as a toxic molecule, we have studied N-formyl-methionyl-leucyl-phenylalanine (fMLF) -stimulated responses of human neutrophils (PMNs) during various conditions of NO modulation in unprimed and bacterial lipopolysaccharide (LPS) -primed cells. Effects of various NO modulators were assessed on stimulated superoxide (O2-) generation, granule exocytosis, homotypic aggregation, and rises in intracellular free Ca2+ ([Ca2+]i). Significant differences in the effects of various NO modulators on inflammatory responses of PMNs kept in stirred suspension versus those kept under static and/or adherent conditions, were observed. L-arginine, the physiological substrate for NO synthase (NOS), and NG-nitro-L-arginine methyl ester, an inhibitor of NOS, both caused a 40-50% inhibition of LPS-induced priming of O2- generation in PMNs in stirred suspension, but not in LPS-primed PMNs under static or adherent conditions. The NO donors, sodium nit...
NO Modulates NADPH Oxidase Function Via Heme Oxygenase-1 in Human Endothelial Cells
Hypertension, 2006
NO is known to induce expression of heme oxygenase-1, an antioxidant enzyme in blood vessels. We tested whether NO might modulate the endothelial NADPH oxidase function via heme oxygenase-1. In human microvascular endothelial cells, the NO donor DETA-NONOate (0.1 to 1 mmol/L) strongly induced expression of heme oxygenase-1 but not Cu/Zn superoxide dismutase. This was associated with a reduction of the superoxide-generating capacity of NADPH oxidase, an effect that depended on de novo gene transcription and heme oxygenase-1 activity. Activation of NADPH oxidase by tumor necrosis factor (TNF) α increased generation of reactive oxygen species. DETA-NONOate alone had little effect on TNF-stimulated reactive oxygen species, but it enhanced the TNF response when: (1) heme oxygenase-1 expression was blocked with specific small-interfering RNA; (2) heme oxygenase-1 activity was blocked by zinc-protoporphyrin; or (3) NADPH oxidase activity was blocked by diphenyleneiodonium. Moreover, the he...
Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species
Proceedings of the National Academy of Sciences, 1993
Nitric oxide, NO, which is generated by various components of the immune system, has been presumed to be cytotoxic. However, NO has been proposed to be protective against cellular damage resulting during ischemia reperfusion. Along with NO there is often concomitant formation of superoxide/hydrogen peroxide, and hence a synergistic relationship between the cytotoxic effects of nitric oxide and these active oxygen species is frequently assumed. To study more carefully the potential synergy between NO and active oxygen species in mammalian cell cytotoxicity, we utilized either hypoxanthine/xanthine cell cytotoxicity, we utilized either hypoxanthine/xanthine oxidase (a system that generates superoxide/hydrogen peroxide) or hydrogen peroxide itself. NO generation was accomplished by the use of a class of compounds known as "NONOates," which release NO at ambient temperatures without the requirement of enzyme activation or biotransformation. When Chinese hamster lung fibroblast...
An integrated approach to assessing nitroso-redox balance in systemic inflammation
Free radical biology & medicine, 2011
Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.