Superoxide Synthesis Research Articles

Diphenylene iodonium sensitivity of a solubilized membrane enzyme from rose cells

An NADH-specific oxidation reduction enzyme has been partially purified from rose cell microsomes by aqueous two-phase partitioning, ultracentrifugation, and ion-exchange chromatography, on the basis of the enzyme's ability to activate lucigenin chemiluminescence in the presence of NADH. The enzyme showed strong similarity to a plasma membrane NADH oxidase (“superoxide synthase” as assayed by lucigenin chemiluminescence; T. M. Murphy and C.-K. Auh, Plant Physiol. 110: 621–629, 1996) in its response to substrate, to Triton X-100, and to diphenylene iodonium, an inhibitor of mammalian neutrophil NADPH oxidase and other flavoenzymes. However, its fluorescence spectrum was not characteristic of flavins and instead was similar to that of pterins. Thus inhibition of an enzyme-catalyzed reaction by diphenylene iodonium does not necessarily imply that the reaction is catalyzed by NADPH oxidase or another flavoenzyme. Superoxide synthesis catalyzed by the enzyme preparation was very low but could be increased at least twofold by the addition of a quinone, menadione. This suggests the enzyme acting in conjunction with a natural quinone could produce activated oxygen species in stressed plant cells.

The free oxygen radical scavenging enzymes and redox status in roots and leaves of Populus x Euramericana in response to osmotic stress, desiccation and rehydration

Summary The redox status (ascorbate, glutathione under reduced and oxidized forms) and the activities of antioxidative enzymes (superoxide dismutase, catalase, ascorbate peroxidase, dehydroascorbate reductase, glutathione reductase) were measured in leaves and roots of Populus euramericana cv. Luisa Avanzo cuttings exposed to wilting or to osmotic stress. Osmotic stress was applied for 12 h with 100 mmol/L mannitol; the wilting state (85% of the original fresh weight), which was reached in 15 min in roots, 75 min in old leaves and 145 min in young leaves, was maintained for 12 h. Superoxide synthesis was drastically enhanced in wilted organs vs. osmotically stressed ones. Wilted and osmotically stressed roots exhibited a lack of stress-inducible antioxidative enzymes and a decrease in glutathione and ascorbate contents with the duration of treatments. An enhanced capability to dismutate superoxide was observed in wilted leaves and in osmotically stressed old leaves; ascorbate peroxidase activity was solely enhanced in wilted leaves, but ascorbate content remained stable both in the case of desiccated and osmotically stressed leaves. Although glutathione reductase activity decreased in wilted and mannitol-treated leaves, glutathione content remained also stable in stressed leaves. After rehydration of 12-h wilted leaves and roots, no recovery of the initial rate of each enzyme activity was observed. The adaptive response of roots and leaves of poplar to oxidative stress generated by wilting and osmotic stress was discussed.

Does nitric oxide synthase catalyze the synthesis of superoxide?

Does nitric oxide synthase catalyze the synthesis of superoxide?

Chromatographic Comparison of the UVA Sensitizers Present in Brunescent Cataracts and in Calf Lens Proteins Ascorbylated in vitro

The water-insoluble (WI) fraction from aged human lenses contains yellow chromophoric sensitizers, which generate reactive oxygen species (ROS) when irradiated with UVA light. The WI proteins from type I to V brunescent cataract lenses were assayed for UVA-dependent superoxide anion synthesis. Rates varied from 8.4–15 nMol h−1mg protein−1, but there was no significant difference in specific activity between cataract types. When calf lens soluble proteins were incubated with ascorbic acid for 4 weeks and dialyzed, they were capable of generating 30–40 nMol h−1mg protein−1superoxide anion when irradiated with UVA light. Two preparations each of brunescent cataract WI proteins and bovine lens proteins ascorbylated in vitro were extensively digested with proteolytic enzymes and the released amino acids separated by normal phase HPLC. The elution profiles of the digests were very similar based upon the absorbance at 330 nm and fluorescence at 350 nm excitation/450 nm emission. Each peak was pooled and analyzed for the UVA-dependent generation of both superoxide anion and singlet oxygen. Every peak exhibited sensitizer activity, and the UVA-dependent ROS generation was roughly proportional to the absorbance at 330 nm. In addition, the ratio of superoxide anion to singlet oxygen generated was similar with both preparations. These data argue that it is the brown, fluorescent compounds which accumulate during aging and cataract formation that are responsible for the UVA-dependent ROS formation, and that these browning products may be similar to the advanced glycation endproducts produced by ascorbylation of lens proteins under oxidative conditions. This work also presents an initial report of a chromatographic method to separate the UVA-sensitizers present in each of these protein preparations without the use of acid or base hydrolysis.

Coenzyme Q versus hypertension: does CoQ decrease endothelial superoxide generation?

Reports from several research groups – including two small double-blind clinical studies – indicate that supplemental coenzyme Q10(CoQ) is moderately effective as a treatment for hypertension, in humans and in animals. Its efficacy is associated with a decrease in total peripheral resistance, and appears to reflect a direct impact of CoQ on the vascular wall. A reasonable interpretation of these findings is that CoQ is acting as an antagonist of vascular superoxide – either scavenging it, or suppressing its synthesis. By improving the efficiency of shuttle mechanisms that transfer high-energy electrons from the cytoplasm to the mitochondrial respiratory chain, CoQ may decrease cytoplasmic NADH levels and thereby diminish the reductive power that drives superoxide synthesis in endothelium and vascular smooth muscle. If CoQ therapy does indeed lower vascular superoxide levels, it can be expected to decrease the atherothrombotic risk associated with hypertension, and may have broader utility in the management of disorders characterized by endotheliopathy.

The Role of Protein Kinase C and [Ca2+]i in Superoxide Anion Synthesis and Myeloperoxidase Degranulation of Human Neutrophils.

A chemiluminescence procedure has been developed to determine superoxide anion (O2-) generation and myeloperoxidase (MPO) release from human activated neutrophils. By using this procedure, the role of protein kinase C (PKC) and cytosolic calcium ion (Ca2+[i) for O2- generation and MPO release was examined. Activation of Fc gamma R on neutrophils with IgG-coated zymosan (IgGZ) caused a transient rise of Ca2+[i, followed by O2- generation and MPO release. A PKC inhibitor suppressed completely the O2- generation and slightly the MPO release. Direct activation of PKC by a specific PKC activator, phorbol myristate acetate (PMA), induced a remarkable O2- generation and a small MPO release, indicating that PKC may regulate entirely O2- synthesis and partially MPO degranulation. Influx of extracellular Ca2+ induced by the calcium ionophore A23187 provoked MPO release only. Complete inhibition of this MPO release with a Ca2+/calmodulin (CaM)-coupling inhibitor and a CaM inhibitor provides evidence that Ca2+[i may regulate MPO degranulation through direct activation of CaM, but not PKC. The Ca2+/CaM inhibitors significantly prevented IgGZ-induced O2- generation and MPO release, while they did not affect PMA-induced O2- generation and MPO release. These results suggest that in Fc gamma R-stimulated neutrophils, Ca2+[i activates CaM, which in turn mediates not only activation of PKC-induced O2- synthesis and MPO degranulation, but also MPO degranulation without PKC intermediate.

Laboratory Study: Effect of Allopurinol in the Course of Adriamycin Induced Nephropathy

The role of superoxide in adriamycin-induced nephropathy (single dose; i.v. 3 mg/kg) has been studied by blocking superoxide synthesis through the administration of allopurinol (500 mg/L in drinking water). In Experiment I (El), allopurinol administration was started 3 days prior to nephropathy induction and continued until day 14. In Experiment II (EII) allopurinol administration was started 2 weeks after nephropathy induction and was maintained until the end of the experiment (26 weeks). Affected glomeruli frequency and tubulointerstitial lesion index (TILI) were determined at Weeks 2 and 4 (El) and Week 26 (EII). In El, the 24 h mean proteinuria in the nephrotic control group (NCG-I) differed from that of the treated nephrotic group (TNG-I) at Week 1 (TNG = 33.3 ± 6.39 mg/24 h; NCG = 59.8 ± 6.3 mg/24 h; p < 0.05) and 2 (NCG-I = 80.0 ± 17.5 mg/24h; TNG-I = 49.1 ± 8.4 mg/24 h; p < 0.05). No glo-merular alterations were observed and TILI medians were not different in both nephrotic groups at week 2 (NCG-I = 1 + TNG = I +) and 4 (NCG = 4+; TNG = 4+). In EII, NCG-JI and TNG-Il presented different 24 h proteinuria values only at Week 6, (136.91 ± 22.23 mg/24 h and 72.66 ± 10.72 mg/24 h, respectively; p < 0.05). Between nephrotic groups, there was no statistical difference in the median of affected glomeruli (CNG-II = 56%; TNG-U = 48%) and TILI (NCG-II = 8+; TNG-II = 9+). Thus, allopurinol was associated with a transient reduction in proteinuria and it did not alter the progression of the nephropathy

Cyclosporine increases local glomerular synthesis of reactive oxygen species in rats: effect of vitamin E on cyclosporine nephrotoxicity.

We report an investigation of the effects of cyclosporine (CsA) on kidney function, the glomerular synthesis of reactive oxygen species, the peroxidation of lipids, and the levels of thromboxane B2 (TXB2). The effect of the simultaneous administration of the antioxidant vitamin E (Vit E) and CsA in rats was also evaluated. Adult male Wistar rats were treated for 30 days with CsA (30 mg/kg/day), with Vit E (0.05 mg/ml), with CsA plus Vit E, or with the vehicle used for administration of CsA, namely 12.6% ethanol. CsA induced kidney failure and increased the glomerular synthesis of superoxide anion, H2O2, malonyldialdehyde, and TXB2. Vit E minimized the adverse effects of CsA on kidney function and the glomerular synthesis of these compounds. Our results suggest that the acute decrease in glomerular filtration rate induced by CsA might be mediated by the synthesis of reactive oxygen species and subsequent peroxidation of lipids, which increases the levels of TXB2. Treatment with Vit E prevented these effects, suggesting a possible role for antioxidants in the prevention of CsA nephrotoxicity.

H 2O 2 and NO: redox signals in disease resistance

One of the earliest responses of plants to microbial pathogens is the production of active oxygen species (AOS) such as superoxide (O2−) and hydrogen peroxide (H2O2) (Ref. [ 1 Lamb C. Dixon R.A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 251-275 Crossref PubMed Scopus (2522) Google Scholar ]). This so-called `oxidative burst' (the name was adopted from a similar process in mammalian macrophages) is triggered within minutes of infection. The process is initiated after pathogen recognition and involves a series of signal transduction events that typically include phospholipases, GTP-binding proteins, fluxes in Ca2+ and other ions, kinases and phosphatases[ 2 Yang Y. Shah J. Klessig D.F. Signal perception and transduction in plant defense responses. Genes Dev. 1997; 11: 1621-1639 Crossref PubMed Scopus (532) Google Scholar ]. Studies of various plant–pathogen combinations have revealed a striking correlation between the profile of AOS formation in cell cultures and the outcome of the interaction (resistance or susceptibility) in intact plants. Plant–pathogen interactions that induced a hypersensitive response (HR)—a resistance response of the host characterized by a rapid but strictly localized lesion formation, gave rise to a biphasic and sustained production of AOS, whereas interactions leading to disease provoked a monophasic oxidative burst. Similar AOS responses have now also been reported for intact plants[ 3 Draper J. Salicylate, superoxide synthesis and cell suicide in plant defence. Trends Plant Sci. 1997; 2: 162-165 Abstract Full Text PDF Scopus (227) Google Scholar ]. These observations led to the suggestion that sustained AOS formation is critical for establishing disease resistance during the HR. Consistent with this model, a number of studies revealed that AOS contribute to three major defense features of HR, namely hypersensitive cell death, cell wall strengthening and the activation of transcription-dependent responses against pathogens[ 1 Lamb C. Dixon R.A. The oxidative burst in plant disease resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1997; 48: 251-275 Crossref PubMed Scopus (2522) Google Scholar , 4 Brisson L.F. Tenhaken R. Lamb C. Function of oxidative cross-linking of cell wall structural proteins in plant disease resistance. Plant Cell. 1994; 6: 1703-1712 PubMed Google Scholar , 5 Levine A. et al. H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell. 1994; 79: 583-593 Abstract Full Text PDF PubMed Scopus (2259) Google Scholar , 6 Jabs T. Dietrich R.A. Dangl J.L. Initiation of runaway cell death in an Arabidopsis mutant by extracellular superoxide. Science. 1996; 273: 1853-1856 Crossref PubMed Scopus (677) Google Scholar , 7 Jabs T. et al. Elicitor-stimulated ion fluxes and O2− from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4800-4805 Crossref PubMed Scopus (496) Google Scholar ]. The precise role of AOS formation in disease resistance is nevertheless still controversial. For example, in some plant–pathogen interactions, the oxidative burst appears to be insufficient to trigger hypersensitive cell death[ 7 Jabs T. et al. Elicitor-stimulated ion fluxes and O2− from the oxidative burst are essential components in triggering defense gene activation and phytoalexin synthesis in parsley. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4800-4805 Crossref PubMed Scopus (496) Google Scholar , 8 Glazener J.A. Orlandi E.W. Baker C.J. The active oxygen response of cell suspensions to incompatible bacteria is not sufficient to cause hypersensitive cell death. Plant Physiol. 1996; 110: 759-763 Crossref PubMed Scopus (100) Google Scholar ]. Furthermore, the defense genes that are induced by AOS are also up-regulated by abiotic stress, which may indicate that AOS production is part of a general stress defense pathway (possibly associated with necrosis) rather than of a specific response to pathogens. Is nitric oxide toxic or protective?Beligni et al.Trends in Plant ScienceAugust 01, 1999In Brief Full-Text PDF

The superoxide synthases of rose cells . Comparison Of assays

In an effort to identify the enzymatic mechanism responsible for the synthesis of reactive oxygen species produced during the hypersensitive response, preparations of rose (Rosa damascena) cell plasma membranes, partially solubilized plasma membrane protein, and cytosol were assayed for the NADH- and NADPH-dependent synthesis of superoxide using assays for the reduction of cytochrome c (Cyt c), assays for the reduction of nitroblue tetrazolium, and assays for the chemiluminescence of N,N'-dimethyl-9,9'-biacridium dinitrate (lucigenin). Each assay ascribed the highest activity to a different preparation: the Cyt c assay to cytosol, the nitroblue tetrazolium assay to plasma membrane, and the lucigenin assay to the partially solubilized plasma membrane protein (with NADH). This suggests that no two assays measure the same set of enzymes and that none of the assays is suitable for comparisons of superoxide synthesis among different cell fractions. With the plasma membrane preparation, the presence of large amounts of superoxide-dismutase-insensitive Cyt c reductase confounded attempts to use Cyt c to measure superoxide synthesis. With the partially solubilized membrane protein, direct reduction of lucigenin probably contributed to the chemiluminescence. Superoxide synthesis detected with lucigenin should be confirmed by superoxide-dismutase-sensitive Cyt c reduction.

Nitrotyrosine-protein adducts in hepatic centrilobular areas following toxic doses of acetaminophen in mice.

Treatment of mice with a toxic dose of acetaminophen (300 mg/kg, ip) significantly increased hepatotoxicity at 4 h, as evidenced by histological necrosis in the centrilobular areas of the liver, and increased serum levels of alanine aminotransferase (ALT) (from 8 +/- 1 IU/L in saline-treated mice to 3226 +/- 892 IU/L in the acetaminophen-treated mice). Serum levels of nitrate plus nitrite (a marker of nitric oxide synthesis) were also increased from 62 +/- 8 microM in saline-treated mice to 110 +/- 14 microM in acetaminophen-treated mice (P < 0.05). Regression analysis of serum ALT levels to serum nitrate plus nitrite levels in individual mice revealed a positive, linear relationship between serum ALT levels and serum nitrate plus nitrite levels with a correlation coefficient of 0.9 (P < 0.05). The y intercept value (nitrate plus nitrite level) was 63 +/- 15 microM. Immunohistochemical analysis of liver sections from acetaminophen-intoxicated mice using an anti-3-nitrotyrosine antibody indicated tyrosine nitration in the proteins of the centrilobular cells. Tyrosine nitration has been shown to occur by peroxynitrite, a reactive intermediate formed by an extremely rapid reaction of nitric oxide and superoxide and a species which also has hydroxyl radical-like activity. Analysis of liver sections using an anti-acetaminophen antiserum indicated the centrilobular cells also contained acetaminophen-protein adducts, a reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine residues on proteins. These data are consistent with acetaminophen metabolic activation leading to increased synthesis of nitric oxide and superoxide and to peroxynitrite as an important intermediate in the toxicity.

Crystal structure at 180°K of bis-3,5-diisopropylsalicylatobisdimethylsulfoxidozinc(II) and the inhibition of seizures and polymorphonuclear leukocyte chemiluminescence

Dimethylsulfoxide (DMSO) formed a ternary complex when mixed with a Zn-3,5-diisopropylsalicylate complex of unknown structure. The structure of this new ternary complex was characterized in an initial effort to understand the nature of this compound. Since the original complex is known to have anticonvulsant activity, the new ternary complex was also examined for anticonvulsant activity. The original complex was examined for inhibition of the polymorphonuclear leukocyte (PMNL) respiratory burst in an effort to mechanistically account for zinc complex mediated anticonvulsant activity. Dissolving the structurally unknown complex in DMSO gave crystals of a characterizable complex with an empirical formula C 30H 46O 8S 2Zn. Crystallographic data: P 1 , Z=2, a=8.063(1), b=12.452(2), c=17.951(2) Å, α=74.42(1), β=77.07(1), γ=89.50(1)°. The structure was refined to R=0.03, R w=0.04 for 3815 independent reflections with I > 2 σ( I). This complex is mononuclear, with two 3,5-diisopropylsalicylate ligands and two bonded DMSO ligands, Zn(II)(3,5-DIPS) 2(DMSO) 2. Zn(II) is coordinate covalently bonded to four O atoms in a strongly distorted tetrahedral arrangement. Each DMSO ligates via its sulfoxide O atom while each 3,5-diisopropylsalicylate ligand is monodentate. The non-ligating carbonyl O atom of each 3,5-DIPS is free except for an intramolecular hydrogen bond from the hydroxy group of the same ligand. Both 3,5-DIPS acid and Zn(II)(3,5-DIPS) 2(DMSO) 2 were examined for anticonvulsant activity in the Maximal Electroshock (MES) and Metrazol (MET) models of seizures and found to prevent both types of seizures. The Zn complex was qualitatively and quantitatively more effective than treatment with the free ligand. The influence of a Zn 3,5-DIPS complex and of the ligand 3,5-DIPS on PMNL oxidative metabolism was also studied to help understand the mechanism of anticonvulsant activity of these compounds. A dose-related and significant decrease in chemiluminescent (CL) response to opsonized Zymosan was observed, and the Zn complex was significantly more effective than the free ligand.It is concluded that mononuclear Zn complexes have anticonvulsant activity in Grand Mal and Petit Mal models of seizure possibly due to inhibition of the synthesis of superoxide or down-regulation of Nitric Oxide Synthase in activated phagocytic cells of the central nervous system.

Ethanol Inhibition of Phagocytosis and Superoxide Anion Production by Microglia

Ethanol consumption has been associated with aberrant immune responses resulting in increased susceptibility to infection including opportunistic infections of the central nervous system. We have investigated the effects of chronic ethanol treatment on phagocytosis and production of superoxide anion by microglia. Phagocytosis of radiolabeled opsonized E. coli was markedly suppressed by treating microglia with ethanol. The unstimulated synthesis of superoxide anion was not altered by ethanol treatment of microglia, but ethanol treatment effectively suppressed phorbol-12 myristate-13 acetate-stimulated microglia superoxide anion production. The results indicate that ethanol inhibition of microglia function may play a role in increased susceptibility for central nervous system infections, particularly in immunocompromised subjects.

Autocoid synthesis and action in abnormal placental flows reviewed: Causative vs. compensatory roles

The increased fetal-placental vascular resistance seen in preeclampsia and/or IUGR has been empirically ascribed to deficiencies in synthesis of vasodilator autocoids in this vascular bed. However, abnormal development of the vascular tree and thus increased resistance to flow could lead to alterations in production of autocoids as a compensatory response, particularly as physical factors such as flow/shear stress over endothelial cells are potent stimuli to autocoid synthesis. The nitric oxide radical (NO) is a potent vasodilator of the human fetal-placental vasculature. Alterations in NO synthesis have been reported in the placenta in preeclampsia. The bioactivity of NO is regulated by its interaction with and inactivation by the superoxide anion. However, the product of this interaction, the strong-lived oxidant peroxynitrite, while acting acutely as a vasodilator, acts in a chronic manner to exert deleterious cytotoxic effects and causes vascular dysfunction. Production and action of peroxynitrite can be identified by the presence of nitrotyrosine residues. We have determined the immunohistochemical localization and quantification of the endothelial nitric oxide synthase isoform (eNOS) and of nitrotyrosine residues (NT) in placental villous tissue from normotensive pregnancies in comparison with those complicated by preeclampsia and/or IUGR. Whereas, there was no difference in quantity of eNOS immunostaining in syncytiotrophoblast between the four groups of tissues, more intense eNOS staining was seen in the villous vascular endothelium of preeclamptic and/or IUGR placentae. This parallels our previous observation of increased concentrations of nitrite, a breakdown product of NO, in the umbilical circulation of these pregnancies. No NT immunostaining was seen in trophoblast cells of any placentae or in the vascular endothelium of normotensive placentae. However, significantly more intense NT immunostaining was apparent in the villous vascular endothelium of the pathologic placentae and diffusing into vascular smooth muscle and stroma. Therefore, the increased vascular resistance and abnormal umbilical blood flow velocity waveforms seen in preeclampsia and/or IUGR cannot simply be ascribed to reduced synthesis and action of vasodilator autocoids in fetal placental vasculature. Rather, increased synthesis of some of these vasodilator autocoids e.g., NO may be occurring in the first instance as an adaptive or compensatory response to increased resistance or to a primary anatomic defect found in this vasculature. The effect of this increased autocoid expression will be to try to vasodilate this vasculature to increase blood flow to the placenta and increase oxygen and nutrient extraction. However, in common with many other conditions of oxidative stress where endothelial dysfunction is seen, concomitant increased synthesis of nitric oxide and superoxide may lead to the formation of the deleterious compound peroxynitrite, which results in vascular damage. The relative rates and sites of synthesis and the opportunity for interaction between these radicals may be key to determining whether potentially beneficial or deleterious effects are seen.

Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium.

A clinically available mixture of hydroxyethylrutosides (HR) was examined as a protector against endothelial cell activation by hypoxia in perfused human umbilical vein. The results showed that 500 micrograms/mL HR totally inhibited the adherence of human unstimulated neutrophils to the endothelium of umbilical vein incubated in hypoxic conditions. This inhibition was confirmed by a morphological study performed by scanning electron microscopy. In addition, neutrophils adherent to the hypoxic umbilical vein endothelium became activated, as evidence by the increased release of superoxide anions and synthesis of leukotriene B4. These processes could also be inhibited by HR. In conclusion, the results of this study suggest that the improvement in venous insufficiency observed clinically with HR could, in part, be the result of their ability to inhibit the recruitment and activation of neutrophils by endothelium activated during blood stasis.

Salicylate, superoxide synthesis and cell suicide in plant defence

Salicylate, superoxide synthesis and cell suicide in plant defence

Nitric oxide attenuates interleukin 2-induced lung injury

The pathogenesis of interleukin (IL) 2-induced vascular leak syndrome may be related to neutrophil-mediated endothelial injury. Nitric oxide inhibits neutrophil superoxide anion synthesis and adherence to endothelial cells. The role of systemic nitric oxide in preventing IL-2-induced lung injury was studied in an experimental model. Sprague-Dawley rats (seven per group) were randomized to control, IL-2 treatment (1 x 10(6) units), and IL-2 with sodium nitroprusside 0.2 mg/kg. Lung injury was measured by estimation of extravascular lung water (wet:dry weight) and bronchoalveolar lavage (BAL) protein concentration, and by histological findings. Neutrophil infiltration was evaluated by measuring myeloperoxidase activity and BAL neutrophil concentration. IL-2 produced significant lung damage characterized by leucocyte sequestration (increased myeloperoxidase and BAL neutrophil concentrations), pulmonary congestion and microvascular protein leakage (increased wet:dry weight ratio and BAL protein concentration). This injury was reduced significantly by the addition of sodium nitroprusside, the nitric oxide donor. Nitric oxide reduces IL-2-induced lung injury.

Involvement of protein phosphatase 2A in PKC-independent pathway of neutrophil superoxide generation by fMLP

We examined the effects of okadaic acid, a protein phosphatase 1 and 2A inhibitor, on superoxide generation in human neutrophils. Superoxide generation induced by fMLP was inhibited by low-dose okadaic acid (10-100 nM), but it had no effect on superoxide synthesis by PMA, and the fMLP-induced rise of the intracellular Ca2+ concentration was not affected by low-dose okadaic acid. These findings suggested that the inhibitory mechanism of okadaic acid might involve PKC-independent and Ca(2+)-independent pathways in fMLP induced NADPH oxidase activation. Both fMLP-stimulated phosphorylation of serine residues in p47phox and its translocation to the plasma membrane were suppressed by low-dose okadaic acid. On the other hand, PMA-induced phosphorylation and translocation of p47phox were not affected by such a low dose of okadaic acid. These findings suggested that fMLP induced phosphorylation of serine residues in p47phox was regulated by protein phosphatase 2A, and its phosphorylation was necessary for translocation and superoxide generation in fMLP-activated human neutrophils.

PGI2 Analogue, sodium beraprost, suppresses superoxide generation in human neutrophils by inhibiting p47PHOX phosphorylation

Sodium beraprost, a newly synthesized PGI2 analogue inhibited in a dose-dependent manner formyl-methionyl-leucyl-phenylalanine (fMLP) induced superoxide generation of human neutrophils, but it had no effect on the superoxide synthesis by phorbol myristate acetate (PMA) or A23187. Sodium beraprost inhibited Ca 2+ influx in fMLP stimulated neutrophils employing fluorometry and confocal microscopy. These findings suggested that the inhibitory effect of sodium beraprost on fMLP induced speroxide generation was due to suppression of Ca 2+ influx. To examine the relationship between the effect of sodium beraprost and phosphorylation of p47phox (the 47kDa cytosolic phagocyte oxidase factor), immuno-precipitation of p47phox and western blotting for phospho-amino acids were performed. Phosphorylation of serine residues of p47phox induced by fMLP was reduced in the presence of sodium beraprost in a dose-dependent manner. The reduction in phosphorylation was accompanied by a reduction in p47phox and p67phox translocation to the plasma membrane and superoxide generation. These findings suggested that p47phox phosphorylation was necessary for translocation and superoxide generation in fMLP activated neutrophils, and that p47phox phosphorylation was regulated by a Ca 2+ dependent mechanism. These observations suggested that sodium beraprost inhibited fMLP induced superoxide generation of human neutrophils by the inhibition of p47phox phosphorylation and translocation by aCa 2+dependent mechanism.

Doxycycline suppression of ischemia-reperfusion-induced hepatic injury

Leukocytes, in particular polymorphonuclear neutrophils (PMNs), are believed to play a central role in ischemia-reperfusion (I/R)-induced tissue injury. Changes in endothelial cells occurring during ischemia promote PMN binding to these cells during reperfusion, which primers PMN synthesis of oxygen radicals and release of cytotoxic proteins. These events lead to vascular damage and subsequent tissue injury. Recently we have shown that doxycycline (Dc), a member of the tetracycline family of antibiotics, inhibits PMN superoxide (O2) synthesis and degranulation in vitro. It also suppresses PMN-mediated RBC, fibroblast, and endothelial cytotoxicity, properties of the drug that may make it of use to protect tissues from I/R-induced injury. In this study we demonstrate that Dc administration either prior to clamping of the portal circulation, or 1 h after the reperfusion, significantly suppressed liver damage as assessed by serum levels of a marker of hepatic injury, alanine aminotransferase (s-ALT). The reduction in s-ALT was not a result of reduced reflow in the Dc-treated rats as indicated by Evans' blue perfusion data. The findings suggest that Dc and possibly other tetracyclines may be of value in protecting tissues and organs from I/R-mediated damage even if the drug is given after the ischemic event has occurred.