NADPH oxidase

The role of NADPH oxidase (NOX) and the regulatory subunit p47phox for hypoosmotic ROS generation was studied in cultured rat astrocytes and brain slices of wilde type and p47phox knock-out mice. Cultured rat astrocytes express mRNAs encoding for the regulatory subunit p47phox, NOX1, 2, and 4, and the dual oxidases (DUOX)1 and 2, but not NOX3. Hypoosmotic (205 mosmol/L) swelling of cultured astrocytes induced a rapid generation of ROS that was accompanied by serine phosphorylation of p47phox and prevented by the NADPH oxidase inhibitor apocynin. Apocynin also impaired the hypoosmotic tyrosine phosphorylation of Src. Both, hypoosmotic ROS generation and p47phox serine phosphorylation were sensitive to the acidic sphingomyelinase inhibitors AY9944 and desipramine, the protein kinase C (PKC)ζ-inhibitory pseudosubstrate peptide, the NMDA receptor antagonist MK-801 and the intracellular Ca2+ chelator BAPTA-AM. Also hypoosmotic exposure of wilde type mouse cortical brain slices increased ROS generation, which was allocated in part to the astrocytes and which was absent in presence of apocynin and in cortical brain slices from p47phox knock-out mice. Also ammonia induced a rapid ROS production in cultured astrocytes and brain slices, which was sensitive to apocynin. The data suggest that astrocyte swelling triggers a p47phox-dependent NADPH oxidase-catalyzed ROS production. The findings further support a close interrelation between osmotic and oxidative stress in astrocytes, which may be relevant to different brain pathologies including hepatic encephalopathy. © 2007 Wiley-Liss, Inc.

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)

An association between excessive zinc (Zn) accumulation in brain and incidences of Parkinson's disease (PD) has been shown in several epidemiological and experimental investigations. The involvement of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and glutathione (GSH) in the pathogenesis of PD has also been proposed in a few studies. Despite the implicated role of oxidative stress in PD, the entire mechanism of Zn-induced dopaminergic neurodegeneration has not yet been clearly understood. The present study aimed to investigate the involvement of NADPH oxidase and GSH in Zn-induced dopaminergic neurodegeneration and also to assess its similarity with paraquat (PQ)-induced rat model of PD. Male Wistar rats were treated either with Zn (20 mg/kg; i.p.) or PQ (5 mg/kg; i.p.) in the presence and absence of NADPH oxidase inhibitor, apocynin (10 mg/kg; i.p.) and a GSH precursor, N-acetyl cysteine (NAC; 200 mg/kg; i.p.) either alone or in combination along with the respective controls. Apocynin and/or NAC pre-treatment significantly alleviated Zn- and PQ-induced changes in neurobehavioral deficits, number of dopaminergic neurons and contents of the striatal dopamine and its metabolites. Apocynin and/or NAC also mitigated Zn- and PQ-induced alterations in oxidative stress, NADPH oxidase activation and cytochrome c release, caspases-9 and -3 activation and CD11b expression. The results obtained thus suggest that Zn induces oxidative stress via the activation of NADPH oxidase and depletion of GSH, which in turn activate the apoptotic machinery leading to dopaminergic neurodegeneration similar to PQ.

Fish macrophage function can be altered after exposure to pathogens as well as to xenobiotics. Considering that wild and farmed fish can be exposed in their habitats simultaneously to different types of stressors, including chemical contaminants (e.g. heavy metals) and pathogens (e.g. bacteria), it is fundamental to study their impact either isolated or in combination. Therefore, the present study aimed to evaluate the effects of copper and bacterial lipopolysaccharide (LPS), alone and in combination, on the transcription of target genes related with immune system, respiratory burst activity and cell death, using rainbow trout macrophages as in vitro model. A cell viability experiment was performed to determine the sub-lethal concentrations of copper for rainbow trout macrophages and the LC50-24 h was estimated at 60 μM. The expression of proinflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumour necrosis factor-α (TNFα) increased after copper and copper plus LPS exposure. Copper and LPS interact positively inducing an increase in cytokine expression, which may be indicative of an increased inflammatory response. However, the increase in TNFα mRNA expression induced by 50 μM copper was not accompanied by protein secretion indicating that mRNA abundance does not always reflect the level of protein and that the translation of the TNFα mRNA is somehow inhibited. Serum amyloid A (SAA) and trout C-polysaccharide binding protein (TCPBP) mRNA expression also increased after copper, LPS or LPS plus copper exposure, indicating a role of acute phase proteins in the local response to inflammation. NADPH oxidase and glutathione peroxidase gene expression increased in macrophages after 24 h exposure to copper, LPS or LPS plus copper. The results from the present study improve the understanding of mechanisms involved in copper toxicity, as well as the interaction with a simulated-inflammatory process.

Objectives: To reduce the increase of oxidative stress and the upregulation of CD40L during stenting procedure using ascorbic acid infusion. Background: CD40L upregulation occurring after coronary Percutaneous Coronary Intervention predicts vascular events but the underlying mechanism is still unclear. Methods: Fifty-six patients undergoing elective coronary stenting were randomly allocated to intravenous infusion of the antioxidant ascorbic acid or placebo. Platelet CD40L and plasma levels of soluble CD40L and of 8-hydroxy-2′-deoxyguanosine, a marker of oxidative stress, were measured before and after coronary stenting. In vitro study was also done to measure reactive oxidant species and CD40L expression in platelets exposed to anoxia-reoxygenation. Results: Placebo-treated patients showed a significant increase of platelet CD40L, soluble CD40L and 8-hydroxy-2′-deoxyguanosine compared to baseline values. Patients given ascorbic acid showed no change of soluble CD40L and platelet CD40L but a significant decrease of 8-hydroxy-2′-deoxyguanosine. After 60 and 120 min, soluble CD40L, platelet CD40L and 8-hydroxy-2′-deoxyguanosine were significantly lower in the ascorbic acid-treated group compared to the placebo-treated one. A significant correlation between platelet CD40L and soluble CD40L and between soluble CD40L and 8-hydroxy-2′-deoxyguanosine was observed. Platelets, in vitro exposed to anoxia-reoxygenation, had a burst of ROS and an upregulation of CD40L that were inhibited by ascorbic acid or apocynin, an inhibitor of NADPH oxidase. Conclusions: This study shows that in patients undergoing coronary stenting CD40L is upregulated with a mechanism which is likely mediated by oxidative stress.

Reactive oxygen species (ROS) are generated by many different cells. Singlet oxygen (1O(2)) and a reaction product of it, excited carbonyls (C=O*), are important ROS. 1O(2) and C=O* are nonradicalic and emit light (one photon/molecule) when returning to ground state oxygen. Especially activated polymorphonuclear neutrophil granulocytes (PMN) produce large amounts of 1O(2). Via activation of the respiratory burst (NADPH oxidase and myeloperoxidase) they synthesize hypochlorite (NaOCl) and chloramines (in particular N-chlorotaurine). Chloramines are selective and stable chemical generators of 1O(2). In the human organism, 1O(2) is both a signal and a weapon with therapeutic potency against very different pathogens, such as microbes, virus, cancer cells and thrombi. Chloramines at blood concentrations between 1 and 2 mmol/L inactivate lipid enveloped virus and chloramines at blood concentrations below 0.5 mmol/L, i.e. at oxidant concentrations that do not affect thrombocytes or hemosta...