Effects of Respiratory Burst Inhibitors on Nitric Oxide Production by Human Neutrophils

Human neutrophils (PMN) activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) simultaneously release nitric oxide (.NO), superoxide anion (O₂-) and its dismutation product, hydrogen peroxide (H₂O₂). To assess whether NO production shares common steps with the activation of the NADPH oxidase, PMN were treated with inhibitors and antagonists of intracellular signaling pathways and subsequently stimulated either with fMLP or with a phorbol ester (PMA). The G-protein inhibitor, pertussis toxin (1-10 μg/ml) decreased H₂O₂ yield without significantly changing. NO production in fMLP-stimulated neutrophils; no effects were observed in PMA-activated cells. The inhibition of tyrosine kinases by genistein (1-25 μg/ml) completely abolished H₂O₂ release by fMLP-activated neutrophils; conversely, NO production increased about 1.5- and 3-fold with fMLP and PMA, respectively. Accordingly, orthovanadate, an inhibitor of phosphotyrosine phosphatase, markedly decreased -NO production and increased O_2;^- release. On the other hand, inhibition of protein kinase C with staurosporine and the use of burst antagonists like adenosine, cholera toxin or dibutyryl-cAMP diminished both H₂O₂ and NO production. The results suggest that the activation of the tyrosine kinase pathway in stimulated human neutrophils controls positively O₂^- and H₂O₂ generation and simultaneously maintains -NO production in low levels. In contrast, activation of protein kinase C is a positive modulator for O_2;^-and *NO production.     

The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Nitric Oxide Reversibly Suppresses Xanthine Oxidase Activity

The effects of nitric oxide (NO) on xanthine oxidase (XOD) activity and the site(s) of the redox center(s) affected were investigated. XOD activity was determined by superoxide (O₂^-) generation and uric acid formation. NO reversibly and dose-dependently suppressed XOD activity in both determination methods. The suppression interval also disclosed a dose-dependent prolongation. The suppression occurred irrespective of the presence or absence of xanthine; indicating that the reaction product of NO and O₂^-, peroxynitrite, is not responsible for the suppression. Application of synthesized peroxynitrite did not affect XOD activity up to 2 μM. Methylene blue, which is an electron acceptor from Fe/S center, prevented the NO-induced inactivation. The results indicate that NO suppresses XOD activity through reversible alteration of the flavin prosthetic site.     

Effects of Superoxide on Nitric Oxide-Dependent N-Nitrosation Reactions

Recent studies have demonstrated that nitric oxide (NO) in the presence of superoxide (O₂^-) may mediate mutagenesis via the N-nitrosation of DNA bases followed by nitrosative deamination to yield their hydroxylated derivatives. We have found that phorbol myristate acetate (PMA)-activated extravasated rat neutrophils (PMNs) will N-nitrosate 2,3-diaminonaphthalene (DAN) to yield its highly fluorescent nitrosation product 2,3- naphthotriazole (triazole) via the L-arginine dependent formation of NO. Addition of SOD enhanced triazole formation suggesting that O₂^- production may inhibit the N-nitrosating activity and thus the mutagenic activity of inflammatory PMNs. The objective of this study was to assess the role of superoxide as a modulator of NO-dependent N-nitrosation reactions using PM A-activated PMNs as well as a chemically defined-system that generates both NO and superoxide. We found that PMA-activation of PMNs reduced the amount of N-nitrosation of DAN by approximately 64% when compared to non- stimulated cells (450 vs. 1250 nM). Addition of SOD but not inactivated SOD or catalase to PMA-activated PMNs enhanced the formation of triazole by approximately 4-fold (1950 nM). In addition, we found that the NO-releasing spermine/NO adduct (Sp/NO; 50μM) which produces approximately 1.0 nmol NO/min generated approximately 8000 nM of triazole whereas the combination of Sp/NO and a superoxide generator (hypoxanthine/xanthine oxidase) that produces approximately 1.0 nmol O₂^-/min reduced triazole formation by 90% (790 nM). Addition of SOD but not catalase restored the N-nitrosating activity. We conclude that equimolar fluxes of superoxide react rapidly with NO to generate products that have only limited ability to N-nitrosate aromatic amino compounds and thus may have limited ability to promote mutagenesis via the nitrosative deamination of DNA bases.     

Spin-Trapping and Chemiluminescence Studies of Neutrophils from a Holstein-Friesian Calf with Bovine Leukocyte Adhesion Deficiency

The ability of neutrophils from a Holstein-Friesian calf with bovine leukocyte adhesion deficiency (the proband with a genetic deficiency of the Mac-1 (CD11b/CD18) glycoprotein corresponding to the receptor of complement iC3b) to generate oxygen radicals was examined using electron spin resonance spectrometry (ESR) combined with a spin-trapping technique and luminol-dependent chemiluminescence spectrometry. When the neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), an ESR spectrum confirming the generation of superoxide anions (O₂^-) was clearly observed in both healthy and diseased calves. However, when the neutrophils were stimulated by opsonized zymosan, appearance of the ESR spectrum was recognized in the healthy calves but not in the diseased calf. Similar results were obtained from chemiluminescence experiments.     

Studies on the Nature and Activation of O2--forming NADPH Oxidase of Leukocytes. Identification of a Phosphorylated Component of the Active Enzyme

Highly active superoxide (O₂^-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b_-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b_-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b_-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b_-245 is involved in the activation mechanism of the O₂^--forming enzyme responsible for the respiratory burst in phagocytes.     

Enhanced Superoxide Production with on Change of the Antioxidant Activity in Gingival Fluid of Patients with Chronic Adult Periodontitis

In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O₂^- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myrystate acetate (PMA) decreased the O₂^- formation in control GCF, while in CAP patients produced a marked enhancement of O₂^- generation.

The circulating PMN of control subjects did not show a spontaneous O₂^- formation, differently from CAP patients. On the contrary, a similar O₂^- production was measured when the circulating PMN were stimulated with PMA.

Moreover, the antioxidant activity measured in 10μl of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O₂^- formation supported by a xanthine/xanthine oxidase system.

Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O₂^- formation in respect to the intrinsic antioxidant property of GS.     

Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H⁺ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.     

Superoxide anion burst and taxol production induced by Ce in suspension cultures of Taxus cuspidata

Generation of reactive oxygen species (ROS) induced by Ce⁴⁺ in suspension cultures of Taxus cuspidata was investigated. The burst of superoxide anions (O₂√⁻) occurred rapidly after the addition of Ce⁴⁺ and reached maximum at 4.3 h, while the total level of the cellular reactive oxygen species maintained unchanged. The intracellular superoxide dismutase (SOD) and catalase (CAT) were activated while the intra/extracellular peroxidases (PODs) were inhibited accompanying the O₂√⁻ burst. The pretreatment of the suspension cultures with diphenylene iodonium (DPI), a suicide inhibitor of the NADPH oxidase, blocked the O₂√⁻ burst, inhibiting the cell apoptosis and taxol production induced by Ce⁴⁺. These results show that NADPH oxidase played a key role in O₂√⁻ burst and O₂√⁻ served as a mediator of Ce⁴⁺ for cell apoptosis and taxol production. The pretreatments of the suspension cultures with anthracene-9-carboxylate, an ion-channel blocker, nifedipine, a Ca²⁺-channel blocker, neomycin, a phospholipase C (PLC) inhibitor, or suramin, a G-protein inhibitor, decreased O₂√⁻ burst induced by Ce⁴⁺. It is thus inferred that Ce⁴⁺-induced O₂√⁻ burst, which mediated cell apoptosis and taxol production by activating the ion-channels, PLC, G-proteins and NADPH oxidase.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils: Effects of recombinant human colony stimulating factors and dihydrocytochalasin B

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O₂ ⁻ production by neutrophils. We confirmed a time dependent augmentation of O₂ ⁻ production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O₂ ⁻ production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O₂⁻-production that was first evident at 2 hours. The fall in O₂⁻-forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O₂⁻, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.     

Direct measurement of oscillatory generation of superoxide anions by single phagocytes

Phagocytic cells such as neutrophils generate superoxide anions (O₂⁻) within phagocytic vacuoles for killing and digesting microorganisms. Here we report the simultaneous observation of morphological changes and O₂⁻ generation in single phagocytic cells during phagocytosis. Point stimulation of a cell by contact with an opsonized microelectrode at the cell surface induced significant deformation to engulf the electrode, and also induced the O₂⁻ generation which was measured by the electrode. Periodic fluctuations in the magnitude of the O₂⁻ generation were observed in the time course. These oscillations may be caused by metabolic regulation of the formation of NADPH, which is the substrate for the O₂⁻ generation.     

Effect of isorhapontigenin on respiratory burst of rat neutrophils

The effect of Isorhapontigenin (Iso) isolated from Belamcanda chinensis on respiratory burst of rat neutrophils was investigated. Iso (1, 10, 100 mmol/l) showed an inhibitory effect on superoxide anion and hydrogen peroxide production in phorbol myristate acetate (PMA) activated rat neutrophils in a concentration-dependent manner. Scanning electron microscopy detected that Iso (100 mmol/l) protected against surface changes in rat neutrophils stimulated with PMA. Also, 100 mmol/l Iso inhibited the release of beta-glucuronidase from the activated neutrophils. Electron-spin resonance (ESR) detected that Iso scavenged oxygen free radicals generated in the PMA activated Neutrophils. These results suggest that Iso inhibits respiratory burst of PMA-activated rat neutrophils by scavenging oxygen free radicals.     

The diffusion-controlled reaction of semioxidized tryptophan with the superoxide radical anion

From pulse radiolysis measurements in oxygenated aqueous solution, the semioxidized tryptophan radical (Trp·— formed by the one-electron oxidation of Trp by Br₂^- radical—has been shown to oxidize the superoxide radical anion with a rate constant of k = 2 × 10⁹ M⁻¹ s⁻¹. Proof of this reaction is found in addition of superoxide dismutase (SOD) to the system, which totally eliminates the contribution of the Trp^· + O₂^- mechanism to Trp^· decay. Little, if any, reaction of molecular oxygen with Trp^· may be observed on the time scale of the pulse radiolysis experiment.     

Determination of Manganese Superoxide Dismutase Activity By Direct Spectrophotometry

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O₂^- in a spectrophotometer, is described. Decay of O₂^- generated by KO₂ at pH 9.5, was monitored as the fall in absorbance (A_250nm-A_360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O₂^- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu, Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu, Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.     

铅胁迫下三叶鬼针草内源一氧化氮的生成及其对氧化损伤的缓解效应

对不同浓度铅(Pb)胁迫下三叶鬼针草(Bidens pilosa L.)叶、茎和根中内源一氧化氮(NO)和活性氧(ROS)的生成机制及根系活力的变化,内源NO对Pb胁迫下三叶鬼针草幼苗氧化损伤的缓解效应进行了研究。结果显示,在0~1000 mg/L范围内,随着Pb浓度的增加,叶片中NO含量呈升高趋势,根中NO含量呈先升高后降低的趋势,但仍高于对照,Pb浓度在0~400 mg/L范围内,茎中NO含量与对照持平,Pb浓度大于600 mg/L时,茎中NO含量低于对照;600 mg/L Pb处理能显著增强叶、茎和根中一氧化氮合成酶(NOS)和硝酸还原酶(NR)活性,显著增加叶和茎中亚硝酸根离子(NO_2~-)和类胡萝卜素(Car)含量,NOS、NR、NO_2~-和Car均能促进叶片中内源NO的生成,NOS是根中内源NO生成的主要途径。Pb胁迫使超氧阴离子(O_2~(·-))产生速率、过氧化氢(H_2O_2)含量、丙二醛(MDA)含量和相对电导率(REC)显著升高,从而造成幼苗严重的膜脂过氧化损伤,而胁迫诱发产生的NO能降低根中ROS的产生,促进幼苗根系活力,进而缓解胁迫造成的膜脂过氧化损伤。     

Bleomycin-Iron Damage To Dna With Formation Of 8-Hydroxydeoxyguanosine and Base Propenals. Indications That Xanthine Oxidase Generates Superoxide From Dna Degradation Products

Bleomycin, in the presence of ferric salts, oxygen and a suitable reductant, degrades DNA with the release of base propenals, detected as thiobarbituric acid (TBA) reactivity, and the formation of 8-hydroxydeo-xyguanosine (80HdG) detected by HPLC. When xanthine oxidase is added to the incubated mixture of DNA degradation products, TBA-reactivity is destroyed but 80HdG formation is increased. EPR Spin trapping experiments show that hydroxyl radicals (OH) are formed in the reaction mixture and can be inhibited by the inclusion of either superoxide dismutase or catalase. These findings suggest that the base propenals and possibly malondialdehyde, formed from them, are aldehydic substrates for xanthine oxidase and, the product of this reaction is superoxide (O₂^-) and hydrogen peroxide (H₂O₂). Thus, TBA reactivity is destroyed in the formation of O₂^- and H₂O₂ which stimulate further oxidative damage to DNA resulting in increased 8OHdG formation.     

The Reaction of no With Superoxide

The rate constant for the reaction of NO with ·O₂^- was determined to be (6.7 ± 0.9) × 10⁹ 1 mol^-1 s^-1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O₂^- and the formation of the product ^-OONO. The decay rate of ^-OONO, in the presence of 0.1 moll^-1 formate, ranges from 1.2s^-1 at pH 5 to about 0.2s^-1 in strong base, the latter value probably reflecting catalysis by formate.